Multi-chain eukaryotic display vectors and uses thereof

ABSTRACT

A eukaryotic expression vector capable of displaying a multi-chain polypeptide on the surface of a host cell is provided, such that the biological activity of the multi-chain polypeptide is exhibited at the surface of the host cell. Such a vector allows for the display of complex biologically active polypeptides, e.g., biologically active multi-chain polypeptides such as immunoglobulin Fab fragments. The present invention describes and enables the successful display of a multi-chain polypeptide on the surface of a eukaryotic host cell. Preferred vectors are described for expressing the chains of a multi-chain polypeptide in a host cell separately and independently (e.g., under separate vector control elements, and/or on separate expression vectors, thus forming a matched vector set). The use of such matched vector sets provides flexibility and versatility in the generation of eukaryotic display libraries, for example the ability to generate and to display multi-chain polypeptides by combining and recombining vectors that express variegations of the individual chains of a multi-chain polypeptide. Entire repertoires of novel chain combinations can be devised using such vector sets.

RELATED APPLICATION

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/326,320, filed Oct. 1, 2001.

[0002] The entire teachings of the above application are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0003] The development of phage display technology, whereby non-native(heterologous) polypeptides or proteins are expressed and anchored onthe surface (“displayed”) of a bacteriophage, is a powerful tool foridentifying molecules possessing biological activities of interest, forexample, peptide ligands that bind with high specificity and/or affinityto a given target molecule. Libraries of synthetic oligonucleotides canbe cloned in frame into the coding sequence of genes encoding a phagesurface protein, for example gene III or gene VIII of phage M13. Theseclones, when expressed, are “displayed” on the phage surface as aplurality, due to the variation in sequence of the oligonucleotidesused, of peptide-capsid fusion proteins. These peptide display librariesare then screened for binding to target molecules, usually by affinityselection or “biopanning” (Ladner, R. et al., 1993; Kay et al., 1996;Hoogenboom, H. et al., 1997).

[0004] Phage display library screening is highly advantageous over otherscreening methods due to the vast number of different polypeptides(typically exceeding 1×10⁹) that can be contained in a single phagedisplay library. This allows for the screening of a highly diverselibrary in a single screening step. Display of small peptides or singlechain proteins on phage is advantageous as long as intracellularprocessing or post-translational modification (of which phage orprokaryotic hosts are not capable) are not necessary or desired. Forexample, effective display of a heterologous polypeptide may requirevarious post-translational modifications, intracellular structures, anda compliment of specialized enzymes and chaperone proteins that arenecessary to transport, to glycosylate, to conform, to assemble, and toanchor the display polypeptide properly on the surface of the host cell;however, none of these processes can be accomplished by bacteriophage orprokaryotic cell processes.

[0005] For the display of more complex eukaryotic proteins, for examplemulti-chain polypeptides including immunoglobulins and functionalfragments thereof (e.g., Fabs), or the extracellular domains of MHCmolecules or T cell receptor molecules, there are additional problems toovercome: coordinated expression of the component chains at the levelsof expression sufficient to produce multi-chain products, transport andsecretion of each chain while still accomplishing association into afunctional multi-chain polypeptide, and immobilization (anchoring) of atleast one chain of the multi-chain polypeptide at the host cell surface(i.e., for display), while retaining the proper assembly andfunctionality outside the host cell of the multi-chain polypeptideproduct.

[0006] Display systems utilizing eukaryotic cells, such as yeast, havebeen reported for expressing and displaying single chain polypeptides(Boder, E. and Wittrup, K., 1998; Horwitz, A. et al., 1988; Kieke, M. etal., 1997; Kieke, M. et al., 1999; WO 94/18330; WO 99/36569), howeverthe need exists for improved eukaryotic systems for the expression andfunctional display of multi-chain polypeptides, particularlyimmunoglobulins and fragments thereof. Moreover, there is a need in theart for polypeptide display in a system that harnesses the power ofphage display and the processing advantages of eukaryotic host cells.For example, in contrast to phage display libraries, the maximumpractical size, or “diversity”, of a library that can be expressed inand displayed on the surface of a eukaryotic host cell is about 10⁶ to10⁷.

[0007] These and other technical problems have obstructed the advance ofbiological tools and techniques useful for identifying novel molecules,which possess biological activities of interest. Because of thesetechnical problems, there has been no report to date of materials ormethods for the successful construction of a multi-chain eukaryoticdisplay vector, of the successful display of a multi-chain polypeptide(such as an antibody or a Fab fragment) on the surface of a eukaryotichost cell (such as yeast), of the creation of a multi-chain polypeptidedisplay library in eukaryotic host cells, or of the successful use ofsuch libraries to detect and to isolate specific multi-chainpolypeptides of interest (for example, on the basis of bindingspecificity or affinity for a target molecule).

SUMMARY OF THE INVENTION

[0008] These and other deficiencies in the art are overcome by theinvention described herein, which provides improved display vectors,cells containing display libraries, and methods for the use of suchlibraries and vectors. Specifically, the present invention provides aeukaryotic expression vector capable of displaying a multi-chainpolypeptide on the surface of a host cell such that a biologicalactivity of the multi-chain polypeptide is exhibited at the surface ofthe host cell. Such a vector allows for the display of more complexbiologically active polypeptides, e.g., biologically active multi-chainpolypeptides, than can be obtained via conventional phage displaytechnology.

[0009] The present invention relates to the display and isolation ofbiologically active polypeptides. Specifically, the present invention isdirected to the design and use of novel multi-chain display vectors.

[0010] The present invention describes and enables the successfuldisplay of a multi-chain polypeptide on the surface of a eukaryotic hostcell. Preferred vectors are described for expressing the chains of amulti-chain polypeptide in a host cell separately and independently(e.g., under separate vector control elements, and/or on separateexpression vectors, thus forming a matched vector set). The use of suchmatched vector sets provides a level of flexibility and versatility inthe generation of display libraries, for example the ability to generateand to display multi-chain polypeptides by combining and recombiningvectors that express a variety of the individual chains of a multi-chainpolypeptide. Entire repertoires of novel chain combinations can bedevised using such vector sets.

[0011] The invention further provides the ability to combine the powerof phage display technology (with its ease of manipulation and magnitudeof diversity) with the potential complexity and versatility of amulti-chain eukaryotic display vector (or vector set). The particularmethods described herein permit a practitioner to efficiently transfersequence information of a peptide library (or selected members of thelibrary) between phage display and eukaryotic display systems,accomplished either through the physical transfer of the sequenceinformation from one display vector to the other (using conventionalgenetic engineering techniques) or through the use of a novel dualdisplay vector, operable in both eukaryotic display systems and phagedisplay systems (which necessarily involve prokaryotic expression).

[0012] The present invention is directed to a novel vector, useful in aeukaryotic host cell to display a multi-chain polypeptide on the surfaceof the host cell such that a biological activity of the multi-chainpolypeptide is exhibited at the surface of the host cell, e.g., thebinding activity of a multi-chain polypeptide. Although one preferredembodiment of the vector of the present invention is that of a singlereplicable genetic package, the multi-chain eukaryotic display vectorcan exist as a single vector or as multiple independent vectors of avector set. As used herein, “vector” refers to either a single vectormolecule or a vector set. In one embodiment, the display vector is ashuttle vector, or more precisely a dual display vector, wherein thevector is capable of displaying a biologically active multi-chainpolypeptide on the surface of a eukaryotic host cell transformed withthat vector, or on the surface of a bacteriophage generated as a resultof prokaryotic expression. In another aspect of the invention, thevector can exist as a vector set, wherein each chain of a multi-chainpolypeptide is encoded on one of a matched pair of vectors such thatwhen the vector pair is present in a single eukaryotic cell, the chainsof the multi-chain polypeptide associate and the biological activity ofthe multi-chain polypeptide is exhibited at the surface of theeukaryotic cell.

[0013] The eukaryotic multi-chain display vector of the presentinvention comprises polynucleotides that encode polypeptide chains ofthe multi-chain polypeptide. A first polynucleotide encodes a firstchain of the multi-chain polypeptide linked to an anchor protein. Otherpolynucleotides of the vector (or vector set) encode other chains of themulti-chain polypeptide. All of the polynucleotides of the displayvector(s) are operably-situated in the display vector such that a hosteukaryotic cell, transformed with the vector (or vector set), displaysthe multi-chain polypeptide on the surface of the host cell such thatthe biological activity of the multi-chain polypeptide is exhibited atthe surface of the cell.

[0014] Preferably, the multi-chain polypeptide encoded by themulti-chain display vector(s) of the present invention exists as eithera two-, three-, four-, or multi-chain polypeptide. More preferably, themulti-chain polypeptide is a two-chain or four-chain polypeptidecomprised of two different chains. More preferably, the multi-chainpolypeptide is selected from a group of multi-chain polypeptidesconsisting of T cell receptors, MHC class I molecules, MHC class IImolecules, and immunoglobulin Fab fragments. More preferably, themulti-chain polypeptide is an IgA, IgD, IgE, IgG, IgM, or biologicallyactive fragment thereof. Most preferably, the multi-chain polypeptide isa Fab fragment, wherein the first polynucleotide of the multi-chaindisplay vector comprises a segment that encodes the V_(H) and C_(H)1domains of an Ig heavy chain, and a second polynucleotide comprises asegment that encodes an Ig light chain (V_(L) and C_(L) domains).

[0015] According to the present invention, a first polynucleotideencoding a first chain of the multi-chain polypeptide is linked to ananchor protein. Preferably, the anchor protein is a cell surface proteinof a eukaryotic cell or a functional fragment thereof. More preferably,the anchor protein is α-agglutinin, a-agglutinin, Aga1p, Aga2p, or FLO1.As disclosed herein, linkage of the first chain polypeptide to an anchorprotein can be achieved by a variety of molecular biology techniques.Preferably, the first polynucleotide encoding a first chain of themulti-chain polypeptide is expressed in a eukaryotic host cell as afirst chain-anchor fusion protein; most preferably a first chain:Aga2pfusion protein.

[0016] In one embodiment, one or more of the chains of the multi-chainpolypeptide expressed by the vector(s) in a host cell is linked to areporter gene or tag. Preferably, the tag is an epitope tag selectedfrom the group consisting of 6×His tag, HA tag, and myc tag. Mostpreferably, each chain of the multi-chain polypeptide is linked to adifferent tag.

[0017] Preferably, the multi-chain display vector(s) of the presentinvention provide cloning sites to facilitate transfer of thepolynucleotide sequence(s) that encode the chains of the multi-chainpolypeptide. Such cloning sites comprise restriction endonucleaserecognition site (i.e., restriction sites) positioned to facilitateexcision and insertion of polynucleotides that encode one or more chainsof a multi-chain polypeptide. For example, restriction sites arepreferably located at the 5′ and 3′ ends of the polynucleotide(s) thatencode the chains of the multi-chain polypeptide. The vector of thepresent invention can contain only two restriction sites positioned atthe ends of the polynucleotide segment that includes all segmentsencoding the chains of the multi-chain polypeptide, or, preferably,restriction sites occur at the ends of each polynucleotide segmentencoding a chain of the multi-chain polypeptide (FIGS. 1 and 2).Preferably, each restriction endonuclease recognition site is a uniquerecognition site in the vector.

[0018] The vector (or vector set) of the present invention can beoperable in a variety of eukaryotic host cells, and optionally can beoperable in prokaryotic cells (e.g., bacteria). Preferably, themulti-chain display vector of the present invention is an animal celldisplay vector, a plant cell display vector, a fungus cell displayvector, or a protist cell display vector. More preferably, the displayvector is a yeast display vector. Most preferably, the yeast displayvector is operable in Saccharomyces cerevisiae.

[0019] In another embodiment, the invention is directed to a method forusing the vector (or vector set) described and taught herein fordisplaying a multi-chain polypeptide on the surface of a eukaryotic hostcell, wherein the vector (or vector set) is introduced into theeukaryotic cell and the host cell is cultured under conditions suitablefor expression, transportation, and association of the chains of themulti-chain polypeptide such that the biological activity of themulti-chain polypeptide is exhibited at the surface of the host cell. Asdescribed herein, the polynucleotides encoding the chains of themulti-chain polypeptide can be introduced into the host cell via one ormore vectors. The mode of introducing the vector(s) into the host cellincludes any of the methods for introducing genetic material into a cellknown in the art. Preferred modes include such transformation techniquesknown in the art, including but not limited to electroporation,microinjection, viral transfer, ballistic insertion, and the like.

[0020] Another preferred mode for introducing eukaryotic multi-chaindisplay vectors into a host cell includes the fusion of two haploideukaryotic cells, each expressing at least one of the chains of themulti-chain polypeptide, to produce a diploid host cell expressing both(all) chains, such that the biological activity of the multi-chainpolypeptide is exhibited at the surface of the resulting diploid hostcell. For example, each of the two haptoid cells can contain one (ormore) of the vectors of a vector set (as described herein), such thatthe biological activity of the multi-chain polypeptide is exhibited atthe surface of the diploid host cell resulting from the haploid/haploidfusion. Preferably, the haploid host cell pair is of opposite matingtypes, thus facilitating the fusion (“mating”) of the two eukaryotichaploid cells.

[0021] Another object of the invention is directed to a eukaryotic hostcell that exhibits at the surface of the cell the biological activity ofa multi-chain polypeptide. As described herein, the eukaryotic host cellis preferably an animal cell, a plant cell, a fungus cell, or a protistcell. More preferably the eukaryotic host cell is a yeast cell.Preferably, the yeast host cell is selected from the generaSaccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces,Yarrowia, and Candida. Most preferably, the eukaryotic host cell is S.cerevisiae. Eukaryotic host cells of the present invention can be of anygenetic construct but are preferably haploid or diploid.

[0022] One embodiment of the present invention is directed to aeukaryotic haploid cell pair (preferably of opposite mating types)wherein the first haploid cell expresses at least a first polynucleotideencoding a first chain of a biologically active multi-chain polypeptidelinked to an anchor protein, and the second haploid cell expresses atleast a second polynucleotide encoding a second chain of the multi-chainpolypeptide. As discussed above, fusion of this haploid cell pairresults in a diploid cell that exhibits the biological activity of themulti-chain polypeptide at the surface of the cell. The presentinvention is further directed to assemblages of the various embodimentsdescribed herein, which form novel libraries of multi-chain polypeptidesor of the polynucleotides that encode them. Libraries of the presentinvention comprise a plurality of vectors that encode a multi-chainpolypeptide such that the vector is operable in a eukaryotic host cellto direct expression and secretion of the chains of the multi-chainpolypeptide, association of the chains such that the biological activityof the multi-chain polypeptide is constituted, and anchoring of at leastone chain of the multi-chain polypeptide such that the biologicalactivity of the multi-chain polypeptide is exhibited at the surface ofthe eukaryotic host cell. Preferably, the library of the presentinvention is comprised of library members that encode a multiplicity ofdifferent multi-chain polypeptides. Most preferably, the library iscomprised of library members that encode a multiplicity of variantmulti-chain polypeptides (designed and produced by the variegation of amulti-chain polypeptide template). Novel multi-chain library assemblagesof the present invention include vector libraries, vector set libraries,host cell libraries, and host cell pair libraries as described andtaught herein.

[0023] A related aspect of the present invention is directed to a methodfor transferring nucleic acid sequence information encoding abiologically active multi-chain polypeptide between a phage displayvector and a eukaryotic display vector. One transfer method comprisesinserting polynucleotide sequences encoding the chains of a multi-chainpolypeptide obtained from a phage display vector into a eukaryoticmulti-chain display vector as described and taught herein. Transfer ofthe nucleic acid sequence information encoding the chains of amulti-chain polypeptide can occur as a single transfer event, or canoccur as separate and independent transfer events of nucleic acidsequence information encoding each of the chains of the multi-chainpolypeptide. Similarly, the sequence information encoding each of thechains of a multi-chain polypeptide can be transferred from one displayvector or from multiple different display vectors.

[0024] Another method for transferring nucleic acid sequence informationencoding a biologically active multi-chain polypeptide between a phagedisplay vector and a eukaryotic display vector (and converse to thatjust described) comprises inserting polynucleotide sequences encodingthe chains of a multi-chain polypeptides obtained from a eukaryoticmulti-chain display vector as described and taught herein into a phagedisplay vector. The phage display-eukaryotic display transfer process ofthe present invention is bidirectional, i.e., it can occur from phagedisplay vector to eukaryotic display vector or from eukaryotic displayvector to phage display vector.

[0025] The transfer of nucleic acid sequence information between a phagedisplay vector and the eukaryotic vector of the present invention can beachieved by a variety of genetic transfer methods known in the art(e.g., genetic engineering technology such as recombinant DNAtechnology). Preferred modes of transfer include techniques ofrestriction digestion, PCR amplification, or homologous recombination(e.g., see Liu, Q. et al., 2000; Walhout, A. et al., 2000).

[0026] The present invention is also directed to methods for detectingand isolating multi-chain polypeptides that exhibit a biologicalactivity of interest to the practitioner. The methods of the presentinvention permit the detection of desirable interactions betweenmulti-chain polypeptides and another molecular species, preferablyprotein-protein interactions, and more preferably interactions betweenmulti-chain polypeptides and their ligands/substrates (i.e., targetmolecules). Preferably, the nature of this interaction comprises anon-covalent association (i.e., binding) between the molecular species,however the nature of the binding can be transient (e.g.,enzyme-substrate binding) or of high affinity/avidity (e.g., as withaffinity ligands useful in separations, diagnostics, and/ortherapeutics).

[0027] In one embodiment, the method of the present invention is usefulto screen a library of multi-chain polypeptides (displayed on thesurface of a eukaryotic host cell) by detecting those members of thelibrary that exhibit a biological activity of interest to thepractitioner. In a particularly preferred embodiment, host cells, whichdisplay multi-chain polypeptides exhibiting the biological activity ofinterest, are isolated. Isolated host cells can then, optionally,undergo repeated rounds of screening, or otherwise be manipulated tocharacterize or to utilize the polypeptide sequence of the displayedmulti-chain polypeptide. In addition, the screening method of thepresent invention can be combined with a (preliminary) phage displayscreen and transfer of the selected phage display isolates to theeukaryotic display system described herein for eukaryotic displayscreening.

[0028] In a further embodiment of the present invention, a library ofmulti-chain polypeptides displayed on the surface of a diploideukaryotic host cell, wherein the diploid cell contains a multi-chainvector set as described and taught herein, can be screened to detect(and optionally to isolate) multi-chain polypeptides that exhibit abiological activity of interest to the practitioner. Preferably, thediploid eukaryotic host cell is the product of the fusion of a haploideukaryotic host cell pair as described and taught herein. In oneparticularly preferred embodiment, screened diploid cells displaying amulti-chain polypeptide exhibiting a biological activity of interest canbe isolated and then, optionally, undergo meiosis, whereby the daughter(haploid) cells express separate chains of the selected multi-chainpolypeptide. Daughter cells can then, optionally, be fused with otherhaploid cells that express chains of a multi-chain polypeptide (e.g.,other daughter cells from the same sub-population of isolated diploidcells), producing a recombination population of diploid eukaryotic hostcells that display a multi-chain polypeptide on their surface.Additional rounds of screening and repeat recombination of theindividual chains of the selected multi-chain polypeptide can beperformed, and ultimately the polypeptide sequence of the displayedmulti-chain polypeptide can be characterized or utilized as discussedabove. Recombination of the selected haploid daughter cells can also berecombined (via cellular fusion) with other biased or non-biasedeukaryotic display vectors to produce novel multi-chain display hostcell libraries.

[0029] The eukaryotic display vector can be used to create a eukaryoticdisplay library, such as a yeast display library, comprising a pluralityof such eukaryotic display vectors. Preferably a plurality of eukaryoticdisplay vectors will encode a heterogeneous population of multi-chainpolypeptides, yielding a displayed repertoire of multi-chainpolypeptides, e.g., at least 10⁴, preferably at least 10⁵, morepreferably at least 10⁶, more preferably at least 10⁷, more preferablyat least 10⁸, most preferably at least 10⁹ different polypeptides.

[0030] In particular embodiments of the invention, the anchor is apolypeptide operable as an anchor on the surface of a eukaryotic celland operable as an anchor on the surface of a phage. In otherembodiments, the anchor is a portion of a surface protein that anchorsto the cell surface of a eukaryotic host cell and to the surface of aphage.

[0031] In preferred embodiments of the present invention, the anchor andone chain of the multi-chain polypeptide are expressed as a fusionprotein. In other embodiments, the anchor and one chain of themulti-chain polypeptide become linked on expression via an indirectlinkage, such as, preferably, a Jun/Fos linkage.

[0032] In another embodiment, the invention is directed to a method fordisplaying, on the surface of a eukaryotic host cell, a biologicallyactive multi-chain polypeptide comprising at least two polypeptidechains, comprising the steps of introducing into a eukaryotic host cella first eukaryotic vector comprising a first polynucleotide encoding afirst polypeptide chain of a biologically active multi-chain polypeptidelinked to a cell surface anchor, wherein said vector is operable in aeukaryotic host cell to direct expression and secretion of said firstchain; and a second eukaryotic vector comprising a second polynucleotideencoding a second polypeptide chain of said multi-chain polypeptide,wherein said vector is operable in a eukaryotic host cell to directexpression and secretion of said second chain, wherein a eukaryotic hostcell transformed with said first eukaryotic vector and said secondeukaryotic vector exhibits, on expression of said first and secondpolynucleotides, the biological activity of said multi-chain polypeptideat the surface of the eukaryotic host cell; and culturing said host cellunder conditions suitable for expression of said first and secondpolynucleotides.

[0033] In a further embodiment, the invention is directed to a methodfor displaying, on the surface of a eukaryotic host cell, a biologicallyactive multi-chain polypeptide comprising at least two polypeptidechains, comprising the steps of introducing into a eukaryotic host cella eukaryotic display vector, a eukaryotic display vector set, or a dualdisplay vector as described above, and culturing said host cell underconditions suitable for expression of said polynucleotides.

[0034] The present invention further provides a eukaryotic host cellcomprising a eukaryotic display vector, a eukaryotic display vector set,or a dual display vector as described herein. Suitable eukaryotic hostcells can be animal cells, plant cells, or fungal cells. Preferably, theeukaryotic host cell will be a mammalian cell, an insect cell, and ayeast cell. Most preferably, the eukaryotic host cell will be a yeastcell, e.g., selected from the genus Saccharomyces, Pichia, Hansenula,Schizosaccharomyces, Kluyveromyces, Yarrowia, Debaryomyces, or Candida.Preferred yeast hosts include Saccharomyces cerevisiae, Hansenulapolymorpha, Kluyveromyces lactis, Pichia pastoris, Schizosaccharomycespombe, and Yarrowia lipolytica. The most preferred yeast host cell isSaccharomyces cerevisiae.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The foregoing and other objects, features and advantages of theinvention will be apparent from the following more particulardescription of preferred embodiments of the invention, as illustrated inthe accompanying drawings in which like reference characters refer tothe same parts throughout the different views. The drawings are notnecessarily to scale, emphasis instead being placed upon illustratingthe principles of the invention.

[0036]FIG. 1 is a schematic diagram that illustrates the phagedisplay-eukaryotic display transfer system. The genetic informationencoding the chains of a Fab polypeptide are transferred from a phagedisplay vector to a multi-chain eukaryotic vector of the presentinvention as a single, excised nucleic acid. Unwanted interveninggenetic elements (if any) are then replaced.

[0037]FIG. 2 is a schematic diagram that illustrates the phagedisplay/eukaryotic display transfer system wherein the geneticinformation encoding the chains of a Fab polypeptide are independentlyand separately transferred from a phage display vector to a multi-chaineukaryotic vector of the present invention.

[0038]FIG. 3 is a schematic diagram of the multi-chain yeast displayvector, pTQ3, according to the invention, having unique cloning sitesfor insertion of at least two chains of a multi-chain polypeptide (e.g.,Fab light and heavy chain components), with additional elements arrangedso that the two chains are independently expressed by induction oftandem GALL promoters. In this vector, a first chain (e.g., an Ig lightchain), inserted as an ApaLI/AscI fragment, is expressed as a solublesecretory protein using the Aga2p signal sequence (Aga2p/ss) and fusedwith an HA epitope tag. A second chain (e.g., an Ig heavy chainfragment), inserted as an SfiI/NotI fragment, is expressed as a cellsurface bound fusion protein using the Aga2p/ss and anchoring proteinsubunit (mature Aga2p). The second chain is similarly fused with a mycepitope tag. Other elements useful for plasmid replication (e.g.,pMB1-ori and Cen6/ARSH4) and useful as selective markers (i.e., ampR andTRP) are also indicated.

[0039] FIGS. 4A-4C are representations of data demonstrating independentexpression of fusion proteins. FIG. 4A shows the expression of the 45 kDAga²p-V_(H)-C_(H)1 fusion protein in yeast host cells EBY100 pTQ3-F2 andEBY100 pTQ3-PH1, and FIG. 4B shows the expression of the 30 kDV_(L)-C_(L) chain in yeast host cells EBY100 pTQ3-F2 and EBY100pTQ3-PH1. No fusion products were detected in either empty vectorcontrol. For each host cell, samples were prepared both before (−) andafter (+) galactose induction of the GAL1 promoters operable in theyeast display vectors. FIG. 4C is a representation of immunofluorescencedetection of assembled Fab antibodies on the yeast cell surface. (a)phase contrast (b) detection of HC (c) detection of LC

[0040] FIGS. 5A-C represent a series of cytometric plots. FIG. 5Adepicts yeast cells transformed with pTQ3-F2 (left panel) and pTQ3-PH1(right panel) constructs were left untreated (dotted line) or inducedfor 48 hours at 20° C. (light grey line). Heavy chain (a), light chaindisplay (b) and antigen binding (c) were analyzed using flow cytometry.

[0041]FIG. 6 is a histogram plot illustrating whole cell ELISA of threedifferent anti-streptavidin Fabs displayed on the surface of yeast hostcells EBY100 pTQ3-F2, EBY100 pTQ3-A12, and EBY100 pTQ3-4C8. Antigenbinding, LC display and HC display are indicated respectively.

[0042]FIG. 7 is a cytometric plot of yeast cell mix. EBY100 pTQ3-F2,EBY100 pTQ3-A12, and EBY100 pTQ3-A12/pESC were double-labeled for bothantigen binding and LC display. A plot of LC display against antigenbinding and a gating for normalized antigen binding are indicated.

[0043] FIGS. 8A-8D are representations of data showing binding to yeastrepertoires and individually selected yeast clones at different antigenconcentrations. FIG. 8A shows a series of histograms of antigen bindingand Fab display are shown for the unselected library (a) and polyclonaloutputs of selection round 1, 2 and 3 (b, c, d). The diversifiedanti-streptavidin yeast repertoire was subjected to three rounds ofFACS. The sorting gate used in each library selections is indicated.FIG. 8B shows polyclonal FACS analysis at different antigenconcentrations of a FACS affinity selection campaign of aanti-streptavidin repertoire. A series of bivariant cytometric plotslabeled for both antigen binding and Fab display show an increase in thepopulation of yeast cells showing increased ratio of antigen binding toFab display. FIG. 8C shows data obtained from yeast cells displaying thewild-type F2 (represented by “o”) and mutants R2E10 (represented bytriangles), R3B 1 (represented by squares) and R3H3 (represented bydiamonds) were labeled with anti-HA mAb and streptavidin-PE. The meanfluorescence for streptavidin binding was monitored over time. Thedissociation rate constant is calculated from the slope of the line.FIG. 8D shows a series of cytometric plots of two selection campaignsusing either Kingfisher in combination with FACS (Right column) or FACSalone (right column). The cytometric plots indicate the increasingpercentage of antigen binding cells through unselected (a) round 1 (b)and round 2 (c) of selection.

[0044]FIG. 9 is a schematic diagram of the heavy chain yeast displayvector, pTQ5-HC, according to the invention, having a heavy chainfragment insert under the control of an inducible GAL1 promoter. The Igheavy chain fragment is positioned as a SfiI/NotI insert fragment, andis expressed as a cell surface bound fusion protein using the Aga2psignal sequence (Aga2p/ss) and anchoring protein subunit (Aga2pprotein). The heavy chain fragment (HC) is fused to a myc epitope tag.Other elements necessary for plasmid replication (i.e., pMB 1-ori andCen6/ARSH4), yeast mating (i.e., Matα terminator) and useful asselective markers (i.e., ampR and TRP) are also indicated.

[0045]FIG. 10 is a representation of a western blot demonstratingexpression of the 45 kD Aga2p-HC fusion product as detected with an antic-Myc antibody in the haploid parent yeast cell EBY100 pTQ5-HC (lane 2)compared to the (control) empty vector yeast host cell EBY100 pTQ5(lane 1) and the (standard) Fab display vector yeast host cell EBY100pTQF2 (lane 3).

[0046]FIG. 11 is a series of cytometric plots showing HC display on thesurface of yeast cells without the presence of a light chain at timeequal to zero (i.e., background; solid black lines) and 48 hours afterinduction (dotted lines). Yeast cells EBY100 pTQ5-HC, and control yeastcells EBY100 pTQ5, were labeled with anti-CHI and rabbit anti-mouse IgGFITC to detect the presence of the HC, and also with streptavidin FITC(strep-FITC) to detect antigen binding activity on the yeast surface. HConly can be seen displayed on the yeast cell surface but does not haveany antigen binding activity in the absence of a paired LC.

[0047]FIG. 12 is a schematic diagram of the light chain yeast expressionvector, pTQ6-LC, according to the invention, having a light chain insertunder the control of an inducible GAL1 promoter. The Ig light chain ispositioned as an ApaL1/AscI insert fragment, is expressed as a solubleprotein using the Aga2p/ss. The light chain fragment (LC) is also fusedwith a HA epitope tag. Other elements useful for plasmid replication(e.g., pUC1-ori and Cen6/ARSH4) and useful as selection markers (i.e.,ampR and Blastocidin®) are also indicated.

[0048]FIG. 13 is a representation of a western blot demonstratingexpression of the 60 kD light chain polypeptide as detected in theculture supernatant with an anti-HA antibody in the haploid parent yeastcell W303 pTQ6-LC (lane S2) compared to the (control) empty vector yeasthost cell W303 pYC6 (lane S1).

[0049]FIG. 14 is a histogram plot illustrating whole cell ELISAdetermination of streptavidin binding activity on the cell surface ofparent haploid yeast cells (W303 pTQ6-LC and EBY10O pTQ5-HC) compared tothe derived diploid yeast cell (DIPLOID LC/HC) and control empty vectoryeast host cell W303 pYC6 and standard Fab display vector yeast hostcell EBY100 pTQ3-F2.

[0050] FIGS. 15A-15C are a series of FACS histograms showing antigenbinding and light chain display on an anti streptavidin haploid HCparent (A) and a diploid control containing empty LC and HC expressionplasmids (B) and a positive diploid expressing a streptavidin specificFab on its surface (C).

[0051]FIG. 16 is a representation of a western blot demonstratingexpression of the 30 kD LC polypeptide as detected with an anti-HAantibody in the diploid yeast cell formed by mating EBY100 pTQ5-HC withW303 pTQ6-LC (lane 3) compared to the (control) diploid yeast cellformed by mating EBY100 pTQ5 with W303 pYC6 (lane 2), and the parent LCvector yeast host cell W303 pTQ6-LC (lane 1).

[0052]FIG. 17 is an illustration of a western blot demonstratingexpression of the 45 kD Aga2p-HC fusion product as detected with an antic-Myc antibody in the diploid yeast cell formed by mating EBY100 pTQ5-HCwith W303 pTQ6-LC (lane 5) compared to the (control) diploid yeast cellformed by mating EBY100 pTQ5 with W303 pYC6 (lane 4), the parent HCvector yeast host cell EBY100 pTQ5-HC (lane 3), the standard Fab displayvector yeast host cell EBY100 pTQ3F2 (lane 2), and the (control) emptyvector yeast host cell EBY100 pTQ5 (lane 1).

[0053] FIGS. 18A-18C are representations of immunofluorescence detectionof combinatorially assembled Fab antibodies on the surface of yeastdiploid cells (A) LC display (B) HC display (C) Antigen binding. The toprow shows immunofluorescence and the bottom row shows phase contrast.

DETAILED DESCRIPTION OF THE INVENTION

[0054] A description of preferred embodiments of the invention follows.

[0055] The invention disclosed in the present application describes thefirst demonstration of the successful expression, transport, assembly,and immobilization (or “display”) of a functional heterologousmulti-chain polypeptide (e.g., Fab antibody fragments) on the surface ofa eukaryotic host cell (e.g., yeast). The present invention makespossible the construction of vector libraries and eukaryotic host celllibraries, wherein the cells display a highly variable repertoire ofmulti-chain polypeptides, which multi-chain polypeptides exhibit a highdegree of sequence diversity within the repertoire and a consequentlyhighly variable range of biological activities such as target (e.g.,antigen) specificity. One skilled in the art will appreciate that, byfollowing the teaching of the present invention, a vast array ofmulti-chain molecules can be stably expressed on the surface ofeukaryotic host cells such as yeast.

[0056] Definitions

[0057] Unless otherwise defined herein, the language and terminologyused in the description of the present invention is used in accordancewith the plain meaning of such language and terminology as generallyunderstood and accepted by those of ordinary skill in the art. In anattempt to avoid any latent confusion or ambiguity, particular elementsor features as they relate to the present invention are set forth below.

[0058] As used herein, a “multi-chain polypeptide” refers to afunctional polypeptide comprised of two or more discrete polypeptideelements (i.e., “chains”), covalently or non-covalently linked togetherby molecular association other than by peptide bonding. The chains of amulti-chain polypeptide can be the same or different. A prominentexample of a multi-chain polypeptide is an immunoglobulin (e.g., IgA,IgD, IgE, IgG, and IgM), typically composed of four chains, two heavychains and two light chains, which assemble into a multi-chainpolypeptide in which the chains are linked via several disulfide(covalent) bonds. Active immunoglobulin Fab fragments, involving acombination of a light chain (LC) domain and a heavy chain (HC) domain,form a particularly important class of multi-chain polypeptides. As wellas forming a disulfide bond, the LC and HC of a Fab are also known toeffectively associate (non-covalently) in the absence of any disulfidebridge. Other examples of multi-chain polypeptides include, but are notlimited to, the extracellular domains of T cell receptor (TCR) molecules(involving α and β chains, or γ and δ chains), MHC class I molecules(involving α1, α2, and α3 domains, non-covalently associated to β2microglobulin), and MHC class II molecules (involving α and β chains).Expression of TCR and MHC binding domains in a eukaryotic host cellwhere at least one chain is anchored at the host cell surface with anon-naturally occurring (heterologous) anchor is specificallycontemplated herein.

[0059] The term “biologically active” when referring, e.g., to amulti-chain polypeptide, means that the polypeptide exhibits afunctionality or property that is useful as relating to some biologicalprocess, pathway or reaction. Biological activity can refer to, forexample, an ability to interact or associate with (e.g., bind to)another polypeptide or molecule, or it can refer to an ability tocatalyze or regulate the interaction of other proteins or molecules(e.g., enzymatic reactions). Biological activity can also refer to theability to achieve a physical conformation characteristic of a naturallyoccurring structure, such as the four-chain conformation of naturallyoccurring immunoglobulin gamma (IgG) molecules, the α and β chains of aT cell receptor molecule, or the conformation of an antigen presentingstructure of a major histocompatability complex (e.g., MHC peptidegroove).

[0060] As used herein, “vector” refers to any element capable of servingas a vehicle of genetic transfer, gene expression, or replication orintegration of a foreign polynucleotide in a host cell. A vector can bean artificial chromosome or plasmid, and can be integrated into the hostcell genome or exist as an independent genetic element (e.g., episome,plasmid). A vector can exist as a single polynucleotide or as two ormore separate polynucleotides. A “multi-chain display vector” of thepresent invention is capable, in an appropriate host, of directingexpression of at least one chain of a multi-chain polypeptide andprocessing it for display on the surface of said host. Vectors accordingto the present invention can be single copy vectors or multicopy vectors(indicating the number of copies of the vector typically maintained inthe host cell). Preferred vectors of the present invention include yeastexpression vectors, particularly 2μ vectors and centromere vectors. A“shuttle vector” (or bi-functional vector) is known in the art as anyvector that can replicate in more than one species of organism. Forexample, a shuttle vector that can replicate in both Escherichia coli(E. coli) and Saccharomyces cerevisiae (S. cerevisiae) can beconstructed by linking sequences from an E. coli plasmid with sequencesfrom the yeast 2μ plasmid. A particularly preferred embodiment of thepresent invention is a “dual display vector”, which is a shuttle vectorthat is capable not only of replicating in two different species but iscapable of expressing and displaying heterologous polypeptides in two ormore host species.

[0061] As used herein, “secretion” refers to peptides having a secretionsignal and are processed in the endoplasmic reticulum. If secretedpeptides either contain anchor sequences or associate with the outsideof the cell surface, the peptides are said to be “displayed”. As usedherein, “display” and “surface display” (used interchangeably herein)refer to the phenomenon wherein a heterologous polypeptide is attached,or “anchored”, to the outer surface of a phage or host cell, whereby theanchored polypeptide is exposed to the extracellular environment. Thepresent invention is particularly directed to the display of amulti-chain polypeptide on the surface of a eukaryotic host cell, byexpression of each of the chains in the host cell and the anchoring ofat least one chain of the multi-chain polypeptide to the surface of thehost cell. A “display vector” refers to a vector that is capable ofexpressing a polypeptide in a host cell or phage such that the expressedpolypeptide is displayed on the surface of said host cell or phage.Display vectors of the present invention direct expression ofmulti-chain polypeptides in a host cell or phage such that thebiological activity of the displayed polypeptide is exhibited at thesurface of the host cell or phage. Dual display vectors of thisinvention direct expression of multi-chain polypeptides in at least twodifferent hosts (preferably, e.g., a prokaryotic host cell and aeukaryotic host cell) such that the biological activity of thepolypeptide is exhibited at the surface of the respective hosts.

[0062] The term “repertoire” refers to a population of diversemolecules, e.g., nucleic acid molecules differing in nucleotidesequence, or polypeptides differing in amino aid sequence. According tothe present invention, a repertoire of polypeptides is preferablydesigned to possess a diverse population of molecules that differ intheir binding sites for a target molecule. The polypeptides of therepertoire are designed to have common structural elements, e.g., aswith a repertoire of Fabs, having a well-recognized two-chain structure(Ig light chain associated with V_(H) and C_(H)1 domains of an Ig heavychain) but exhibiting different binding specificities, due to variationin the respective variable regions of the component chains.

[0063] The term “library” refers to a mixture of heterogeneouspolypeptides or polynucleotides. A library is composed of members thathave similar polypeptide or polynucleotide sequences. Where the libraryis a polynucleotide library, it encodes a repertoire of polypeptides(especially, e.g., with regard to the present invention, a repertoire ofmulti-chain polypeptides). Sequence differences between library membersare responsible for the diversity present in the library. The librarycan take the form of a simple mixture of polypeptides orpolynucleotides, or can be in the form organisms or cells, for examplebacteria, viruses, animal or plant cells and the like, that aretransformed with a library of polynucleotides. Where the heterogeneouspolypeptides are expressed and exhibited at the surface of the cells ororganisms forming the library, the library is a “display library”.Advantageously, polynucleotides are incorporated into expressionvectors, in order to allow expression of the polypeptides encoded by thepolynucleotides. In a preferred aspect, therefore, a library can takethe form of a population of host organisms, each organism containing oneor more copies of an expression vector containing a single member of thelibrary in polynucleotide from that can be expressed to produce itscorresponding polypeptide member. Thus, the population of host organismshas the potential to encode a large repertoire of genetically diversepolypeptide variants.

[0064] The present invention is directed to novel multi-chain displayvectors. In one embodiment of the present invention, the polynucleotidesthat encode the chains of the multi-chain polypeptide are present onseparate (i.e., two or more) expression vectors, the compilation ofwhich form a functional display “vector set” (the general term, “vector”encompasses vector sets). For example, if the multi-chain polypeptidewere a two-chain polypeptide comprised of the light chain and the heavychain of a biologically active Fab, the polynucleotide encoding the LCcan be incorporated into one expression vector, and the polynucleotideencoding the HC can be incorporated into a second, separate, expressionvector (most preferably expressed as a HC-anchor fusion protein).Individually, each vector is capable of expressing its respectivepolypeptide chain; the two vectors together form a matched vector set,which set encodes the chains of a biologically active multi-chainpolypeptide. Similarly, separate host cells, each transformed with thedifferent vectors of a vector set, collectively form a matched host cellset (or specifically in the case of a two-vector set, a matched “cellpair”). The vectors and vector sets will preferably also include one ormore selectable markers (e.g., TRP, ampR, and the like) to facilitateselection and propagation of successfully transformed hosts.

[0065] A “host cell” refers to any cell (prokaryote or eukaryote)transformed to contain a vector. According to the present invention,preferred host cells are bacterial cells and eukaryotic cells,including, but not limited to, protist cells, fungus cells, plant cells,and animal cells. Host cells of the invention can be of any geneticconstruct, but are preferably haploid, diploid cells, or multiploid(e.g., as is typical of immortalized cell lines in culture). Preferredhost cells include insect cells (e.g., Sf9), mammalian cells (e.g., CHOcells, COS cells, SP2/0 and NS/0 myeloma cells, human embryonic kidney(HEK 293) cells, baby hamster kidney (BHK) cell, human B cells, humancell line PER.C6TM (Crucell)), seed plant cells, and Ascomycete cells(e.g., Neurospora and yeast cells; particularly yeast of the generaSaccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces,Yarrowia, and Candida). Preferred exemplar yeast species include S.cerevisiae, Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris,Schizosaccharomyces pombe, and Yarrowia lipolytica. A particularlypreferred yeast host cell is S. cerevisiae.

[0066] The term “phage” refers to a “bacteriophage”, which is abacterial virus containing a nucleic acid core and a protectiveproteinaceous shell. The terms “bacteriophage” and “phage” are usedherein interchangeably. Unless otherwise noted, the terms“bacteriophage” and “phage” also encompass “phagemids” (i.e.,bacteriophage the genome of which includes a plasmid that can bepackaged by coinfection of a host with a helper phage). In preferredembodiments of the present invention, the phage is an M13 phage.

[0067] The terms “anchor”, “cell surface anchor” and “anchorpolypeptide”, refer to a polypeptide moiety that, on expression in ahost cell, becomes attached or otherwise associated with the outersurface of the host cell or, in the case of a phage display system, onthe surface of a phage particle (e.g., as part of the capsid or as partof a filament). An anchor polypeptide can be a coat protein moiety, atransmembrane protein moiety, or can be a polypeptide moiety otherwiselinked to the cell surface (e.g., via post-translational modification,such as by a phosphatidyl-inositol or disulfide bridge). The termencompasses native proteins to the host cell or phage, or exogenousproteins introduced for the purpose of anchoring to a host cell wall orphage coat. Anchors include any synthetic modification or truncation ofa naturally occurring anchor that still retains the ability to beattached to the surface of a host cell or phage particle. Preferredanchor protein moieties are contained in, for example, cell surfaceproteins of a eukaryotic cell. Effective anchors include portions of acell surface protein sufficient to provide a surface anchor when fusedto another polypeptide, such as a chain of a multi-chain polypeptide inaccordance with this invention. The use of protein pairs that areseparately encoded and expressed but associate at the surface of a cellby covalent (e.g., disulfide) or non-covalent bonds is also contemplatedas a suitable anchor, and in this regard particular mention is made ofthe yeast α-agglutinin components, Aga1p and Aga2p, which form aglycan-immobilized, disulfide-linked complex on the surface of yeastcells. Another protein pair that can be employed as an anchor areproteins that form “leucine zipper” interactions and the like, such asthe nuclear proteins Jun and Fos (which form a “jun/fos linkage”). Forexample, a display vector can be designed according to this invention todirect the expression in a host cell of a first chain of a multi-chainpolypeptide fused to the leucine zipper moiety of Jun, and a secondvector can be designed to direct independent expression of the leucinezipper moiety of Fos fused to a surface protein of the host. Onexpression of the vector structural genes, the first chain polypeptidewill be associated (i.e., anchored) with the host cell surface via ajun/fos linkage, as the Jun and Fos leucine zipper forms a linkagebetween the first chain polypeptide and the host cell surface proteinfused to the Fos part of the zipper. Any suitable protein binding pairof this sort can be used. Preferred examples of polypeptide anchorsinclude the pIII coat protein of filamentous phage or fragments thereof(e.g., pIII anchor domain or “stump”, see U.S. Pat. No. 5,658,727) forphage display systems, and for yeast display systems FLO1 (a proteinassociated with the flocculation phenotype in S. cerevisiae),α-agglutinin, and a-agglutinin (e.g., Aga1p and Aga2p subunits), andfunctional fragments thereof.

[0068] As used herein, the term “fusion protein” denotes a hybridpolypeptide comprised of amino acid sequences from more than one source,linked together to form a non-naturally occurring, unitary polypeptide.Fusion proteins are prepared, for example, by operably linking codingsequences for the component amino acid sequences in frame, such that,upon expression, they are produced as a single polypeptide.Alternatively, fusion proteins can be assembled synthetically, e.g., bycreating a peptide bond between two or more separate polypeptides.

[0069] As used herein “linked” refers to a functional and structuralconnection between two or more elements. As used herein, the linkedelements typically refer to an operable connection between two or morepolynucleotide elements or polypeptide elements. For example, asdiscussed above, a polypeptide can be linked to an anchor protein (via apeptide bond or via peptide linker), thus forming a fusion protein.Similarly, the polynucleotides encoding the polypeptide and anchorprotein can be linked such that the fusion protein is transcribed andtranslated as a unitary RNA message. Polypeptides can also be indirectlylinked to an anchor via an intermediate association, one example ofwhich is the use of the high-affinity interaction of the Jun and Fosleucine zippers (i.e., a “jun/fos linkage”) to effectively link apolypeptide to the surface of a phage or host cell (Crameri, R. andBlaser, K., 1996). Any suitable heterodimeric or homodimeric pair ofmolecules can be used (Chang, H. et al., 1994; Moll, J. et al., 2001;Pu, W. and Struhl, K., 1993).

[0070] It is understood by persons of ordinary skill in the art thatpolynucleotides, which encode one or more chains of a multi-chainpolypeptide to be expressed and displayed in a phage display or hostcell display system, can be operably linked to a promoter (to facilitatetranscription), or operably linked to a signal sequence or leaderpeptide (to facilitate cellular processing and transport to thesurface). Such genetic control elements and functional linkages theretoare numerous and well known in the art, and the present invention is notlimited by the use thereof. Preferred promoters, however, includeinducible promoters. Particularly preferred promoters (for eukaryoticsystems) include those useful in yeast vectors, such as pGAL1, pGAL1-10,pGa1104, pGa110, pPGK, pCYC1, and pADH1. Other preferred promotersinclude the LacZ promoter (for non-eukaryotic systems). Particularlypreferred signal sequences include the Aga2p signal sequence (foreukaryotic systems), and the pIII signal sequence (for non-eukaryoticsystems).

[0071] Another useful tool known to practitioners in the art, aremolecular labels or “tags” (e.g., epitope tags, reporter genes,radioisotope, fluorescent or chemiluminescent moieties, etc.), whichfacilitate the practitioner's ability. for example, to detect thepresence of a polypeptide linked thereto. Epitope tags (e.g., peptidesegments known to be recognized by particular antibodies or bindingmoieties) are particularly useful herein, in that they can beco-expressed as a fusion partner with one or more chains of amulti-chain polypeptide in a vector or vectors according to theinvention, to permit the detection of expression of one or more chainswith which the tag is co-expressed. As known and used in the art, tagsare typically placed under the same genetic controls as a gene ofinterest (preferably as a component of an expressed fusion protein). Ifand when the gene product of interest is not easily detectable, the tagprovides an easily detectable, and often quantifiable, signal indicatingthe presence of the gene product of interest. By linking a tag to apolypeptide gene product of interest, the practitioner can monitor suchprocesses as, for example, gene expression, polypeptide trafficking,extracellular display, and protein-protein interactions (Fields, S. andStemglanz, R., 1994; Phizicky, E. and Fields, S., 1995).

[0072] Accordingly, the chains of a multi-chain polypeptide can beoptionally linked to one or more tags, either individually or jointly. Avariety of tags are known in the art and are commercially available(Amersham Pharmacia Biotech, Piscataway, N.J.; Applied Biosystems,Foster City, Calif.; Promega, Madison, Wis.; Roche MolecularBiochemicals, Indianapolis, Ind.; Stratagene, La Jolla, Calif.).Preferably, the linkage is achieved via a peptide bond (thus creating afusion protein), wherein the polynucleotide encoding a chain of amulti-chain polypeptide is linked to a tag (e.g., an epitope tag).Preferred tags include polyHis tags, HA tags, and myc tags.

[0073] As used herein, the term “recombinant” is used to describenon-naturally altered or manipulated nucleic acids, host cellstransfected with exogenous nucleic acids, or polypeptides expressednon-naturally, through manipulation of isolated DNA and transformationof host cells. “Recombinant” is a term that specifically encompasses DNAmolecules that have been constructed in vitro using genetic engineeringtechniques, and use of the term “recombinant” as an adjective todescribe a molecule, construct, vector, cell, polypeptide orpolynucleotide specifically excludes naturally occurring molecules.

[0074] Similarly the term “transform” refers generally to any artificial(i.e., practitioncr-controlled) method of introducing genetic materialinto a cell or phage without limitation to the method of insertion.Numerous methods are known in the art and described by the referencescited and incorporated herein. Specifically as applied to the presentinvention, the term “transformant” refers to a host cell that has beentransformed and encompasses, for example, diploid cells, which are theproduct of the controlled fusion of matched haploid cell pairs (as withthe controlled mating of haploid yeast spores of opposite mating type).

[0075] Methods for “transferring” nucleic acid sequence information fromone vector to another is not limiting in the present invention andincludes any of a variety of genetic engineering or recombinant DNAtechniques known in the art. Once again, a vast array of methods areknown in the art and described in the references cited and incorporatedherein. Particularly preferred transfer techniques include, but are notlimited to, restriction digestion and ligation techniques (utilizingunique cloning sites), PCR amplification protocols (utilizing specificprimer sequences), and homologous recombination techniques (utilizingpolynucleotide regions of homology).

[0076] Employing genetic engineering technology necessarily requiresgrowing recombinant host cells (transformants) under a variety ofspecified conditions as determined by the requirements of the organismand the particular cellular state desired by the practitioner. Forexample, the organism can possess (as determined by its geneticdisposition) certain nutritional requirements, or particular resistanceor sensitivity to physical (e.g., temperature) and/or chemical (e.g.,antibiotic) conditions. In addition, specific culture conditions can benecessary to induce or repress the expression of a desired gene (e.g.,the use of inducible promoters), or to initiate a particular cell state(e.g., yeast cell mating or sporulation). These varied conditions andthe requirements to satisfy such conditions are understood andappreciated by practitioners in the art.

[0077] Accordingly, practice of various aspects of the present inventionrequires that host cells be cultured under “conditions suitable” or“conditions sufficient” to achieve or to induce particular cellularstates. Such desirable cellular states include, but are not limited to:cellular growth and reproduction; the expression, secretion ortransport, and association of the chains of a multi-chain polypeptidesuch that the biological activity of the multi-chain polypeptide isexhibited at the surface of the host cell (or phage particle); thefusion of haploid cells to form a diploid cell (e.g., fertilization,zygote formation, the mating of cells of opposite mating types); andmeiosis of a diploid cell to form haploid daughter cells (e.g.,gametogenesis, sporulation). The present invention is not limited by thephysical and chemical parameters of such “suitable conditions”, but suchconditions are determined by the organisms and vectors used to practicethe invention, and by practitioner preference.

[0078] Multi-Chain Polypeptide Eukaryotic Display Vectors

[0079] As outlined earlier, the present invention is directed to a novelgenetic vector, useful in a eukaryotic cell to display a multi-chainpolypeptide on the surface of the cell such that the biological activityof the multi-chain polypeptide is exhibited at the surface of the cell.According to the invention, the multi-chain polypeptide can be encodedin a single vector, or individual chains of the multi-chain polypeptidecan be encoded in a vector set. For example, in one aspect of theinvention, the vector can exist as a vector set, wherein each chain of amulti-chain polypeptide is encoded on one of a matched pair of vectorssuch that when the vector set is present in a single eukaryotic cell,the chains of the multi-chain polypeptide associate at the surface ofthe eukaryotic cell. In another aspect of the invention, the displayvector can be a dual display vector, wherein the vector is capable of(i) expressing in a eukaryotic cell and displaying on the surface of aeukaryotic cell a biologically active multi-chain polypeptide, and (ii)expressing in a prokaryotic cell and displaying on the surface of abacteriophage the biologically active multi-chain polypeptide.

[0080] The multi-chain polypeptide can be any polypeptide comprised oftwo or more discrete polypeptide elements, referred to as chains of themulti-chain polypeptide, which chains are covalently or non-covalentlylinked (other than by peptide bonding) to form a biologically activepolypeptide. Preferably, the multi-chain polypeptide encoded by themulti-chain display vector(s) of the present invention exists as eithera two-, three-, or four-chain polypeptide. The chains of the polypeptidecan be the same (e.g., a homo-dimer, -trimer, or -tetramer) or different(e.g., a hetero-dimer, -trimer, or -tetra-mer). Preferably, themulti-chain polypeptide is a two-chain or four-chain polypeptidecomprised of two different chains. More preferably, the multi-chainpolypeptide is selected from a group of multi-chain polypeptidesconsisting of T cell receptors, MHC class I molecules, MHC class IImolecules, immunoglobulins and biologically active immunoglobulinfragments (e.g., Fabs). More preferably, the multi-chain polypeptide isan IgA, IgD, IgE, IgG, IgM, or biologically active fragment thereof.Most preferably, the multi-chain polypeptide is a Fab fragment of an Ig,wherein the first polynucleotide of the multi-chain display vectorcomprises a segment that encodes the V_(H) and C_(H)1 domains of an Igheavy chain, and a second polynucleotide comprises a segment thatencodes an Ig light chain (i.e., V_(L) and C_(L) domains).

[0081] The chains of the multi-chain polypeptide (e.g., first chain,second chain, third chain, etc.) are encoded as polynucleotides (e.g.,first polynucleotide, second polynucleotide, third polynucleotide, etc.,respectively) in an expression vector. It will be appreciated andunderstood by persons skilled in the art that the polynucleotidesequences encoding the chains do not necessarily have to be insertedinto the identical plasmid, or under the same gene expression control,in order to produce a functional multi-chain polypeptide. For example,the polynucleotide encoding the light chain and heavy chain of an Ig Fabcan be located on separate plasmids and transformed as such into anidentical host cell for co-expression and co-processing into afunctional multi-chain polypeptide.

[0082] It will also be appreciated by those skilled in the art, that thesequences of the polynucleotides that encode the chains of a multi-chainpolypeptide need not originate from an identical, or same source. Forinstance, an Ig molecule can be produced having variable domains (V_(H)and V_(L)) the same as those from a monoclonal antibody having a desiredspecificity, and constant domains (C_(H)1 and C_(L)) from a differentmonoclonal antibody having desired properties (e.g., to provide humancompatibility or to provide a particular complement binding site).

[0083] Moreover, the heterologous polynucleotide encoding the chains ofa multi-chain polypeptide (e.g., Ig domains) can be variegated, toproduce a family of polynucleotide homologs, encoding polypeptide chainsthat vary slightly in amino acid sequence from one another while havingthe same overall structure. In this way, when the homologs areincorporated into different host cells and expressed, a library ofmulti-chain polypeptides of varied sequence are displayed, providing apeptide display library suitable for screening, e.g., to discoverhomologous multi-chain polypeptides having altered biological activity.Such alterations in amino acid sequence can be achieved by suitablemutation or partial synthesis and replacement or partial or completesubstitution of appropriate regions of the corresponding polynucleotidecoding sequences. Substitute constant domain portions can be obtainedfrom compatible recombinant DNA sequences.

[0084] Given proper selection of expression vector components andcompatible host cells, the chains of the multi-chain polypeptide will bedisplayed on the surface of a eukaryotic host cell. Persons skilled inthe art will appreciate that this can be achieved using any of a numberof variable expression vector constructs, and that the present inventionis not limited thereby. The display vector itself can be constructed ormodified from any of a number of genetic vectors and genetic controlelements known in the art and commercially available (e.g., fromInVitrogen (Carlsbad, Calif.); Stratagene (La Jolla, Calif.); AmericanType Culture Collection (Manassas, Va.)). Essentially, the vectorconstruct of the present invention expresses the polypeptide chains foreffective display of a fully assembled, multi-chain polypeptide on thesurface of a eukaryotic cell transformed with the vector such that thebiological activity of the multi-chain polypeptide is exhibited at thesurface of the host cell.

[0085] To achieve effective cellular expression of the multi-chainpolypeptide, the polynucleotides encoding each of the chains of themulti-chain polypeptide are, preferably, linked to a transcriptionalpromoter to regulate expression of the polypeptide chains. The effectivepromoter must be functional in a eukaryotic system, and optionally(particularly in the case of a dual display vector) effective as aprokaryotic promoter as well. In a particular dual display vector, theeukaryotic promoter(s) and the prokaryotic promoter(s) selected forregulating expression of the heterologous polypeptide chains of amulti-chain polypeptide can be the same or different promoters, as longas they are appropriately functional in the intended host organisms.Alternatively, they can be independently selected for the expression ofeach chain in a particular host. The eukaryotic promoter can be aconstitutive promoter but is preferably an inducible promoter. In orderto achieve balanced expression and to ensure simultaneous induction ofexpression, a vector construct that utilizes the same promoter for eachchain is preferred.

[0086] A number of eukaryotic promoters useful in the present inventionare known in the art. Particularly preferred promoters (for eukaryoticsystems) include those useful in yeast expression vectors, such asgalactose inducible promoters, pGAL1, pGAL1-10, pGal4, and pGa110;phosphoglycerate kinase promoter, pPGK; cytochrome c promoter, pCYC1;and alcohol dehydrogenase I promoter, pADH1.

[0087] Preferably, each of the polynucleotides encoding a chain of amulti-chain polypeptide is also linked to a signal sequence (or a leaderpeptide sequence). The signal sequence operates to direct transport(sometimes referred to as secretion) of a nascent polypeptide into oracross a cellular membrane. Chains of a multi-chain polypeptideexpressed in a eukaryotic cell from a vector of the present inventionare transported to the endoplasmic reticulum (ER) for assembly andtransport to the cell surface for extracellular display. An effectivesignal sequence should be functional in a eukaryotic system, andoptionally (particularly in the case of a dual display vector) thesignal sequence should be effective in a prokaryotic system as well.Polynucleotides encoding the chains of a multi-chain polypeptide aretypically directly linked, in frame (either immediately adjacent to thepolynucleotide or optionally linked via a linker or spacer sequence), toa signal sequence, thus generating a polypeptide chain-signal sequencepeptide fusion protein. Preferably, each chain of a multi-chainpolypeptide is fused to a separate signal peptide.

[0088] The signal sequence encoding the signal peptide can be the sameor different for each chain of the multi-chain polypeptide. The signalsequence can be native to the host or heterologous, as long as it isoperable to effect extracellular transport of the polypeptide to whichit is fused. Several signal sequences operable in the present inventionare known to persons skilled in the art (e.g., Mfα1 prepro, Mfα1 pre,acid phosphatase Pho5, Invertase SUC2 signal sequences operable inyeast; pIII, Pe1B, OmpA, PhoA signal sequences operable in E. coli; gp64leader operable in insect cells; IgK leader, honeybee melittin secretionsignal sequences operable in mammalian cells). The signal sequences arepreferably derived from native secretory proteins of the host cell.Particularly preferred eukaryotic signal sequences include those ofα-mating factor of yeast, α-agglutinin of yeast, invertase ofSaccharomyces, inulinase of Kluyveromyces, and most preferably thesignal peptide of the Aga2p subunit of a-agglutinin (especially inembodiments where the anchoring polypeptide to be used is the Aga2ppolypeptide).

[0089] In the particularly preferred embodiment, wherein the multi-chainpolypeptide is a Fab, the first polynucleotide comprises an Aga2p signalsequence in frame with a segment that encodes the V_(H) and C_(H)1regions of an Ig heavy chain, and the second polynucleotide comprises anAga2p signal sequence in frame with a segment that encodes an Ig lightchain.

[0090] The multi-chain eukaryotic display vector of the presentinvention operates in a eukaryotic host cell such that the multi-chainpolypeptide encoded by the vector is displayed on the surface of thehost cell. Anchorage (“tethering” or “display”) on the surface of thehost cell is achieved by linking at least one chain of the multi-chainpolypeptide to a molecular moiety attached to the host cell wall. Morethan one chain of a multi-chain polypeptide can be linked to an anchor,but because the fully assembled multi-chain polypeptide requires (andpreferably contains) only one point of attachment to the host cellsurface, only one chain of the multi-chain polypeptide need be the pointof cellular attachment. Display on the surface of the cell can beachieved by linking at least one of the polypeptide chains to an anchorprotein or functional fragment (moiety) thereof. The effective anchorshould be functional in a eukaryotic system, and optionally(particularly in the case of a dual display vector) the anchor should beeffective as an anchor on the surface of a bacteriophage as well.Preferably, the anchor is a surface-expressed protein native to the hostcell, e.g., either a transmembrane protein or a protein linked to thecell surface via a glycan bridge. Several anchor proteins operable inthe present invention are known to persons skilled in the art (e.g.,pIII, pVI, pVIII, LamB, PhoE, Lpp-OmpA, Flagellin (FliC), or at leastthe transmembrane portions thereof, operable in prokaryotes/phage;platelet-derived growth factor receptor (PDGFR) transmembrane domain,glycosylphosphatidylinositol (GPI) anchors, operable in mammalian cells;gp64 anchor in insect cells, and the like). Preferably, where yeast isthe host, the anchor protein is α-agglutinin, a-agglutinin (havingsubcomponents Aga1p and Aga2p), or FLO1, which naturally form a linkageto the yeast cell surface.

[0091] Linkage of a polypeptide chain to an anchor can be achieved,directly or indirectly, by a variety of molecular biology techniques.The present invention is not limited by the method of chain-anchorlinkage, only by the functional requirement that the linked polypeptidechain is immobilized on the surface of the host cell (or optionallybacteriophage) as a result of such linkage.

[0092] A preferred method of chain-anchor linkage is through theconstruction of a chain-anchor fusion protein. Similar to, andpreferably in concert with, a chain-signal peptide fusion protein, apolynucleotide encoding a chain of a multi-chain polypeptide is directlylinked, in frame (either immediately adjacent to the polynucleotide oroptionally linked via a linker or spacer sequence), to an anchor, thusgenerating a signal peptide-polypeptide chain-anchor fusion protein.

[0093] Alternative modes of peptide-peptide linkage are know in the artand available to achieve the effective chain-anchor linkage of thepresent invention. For example, and as previously cited, a chain of themulti-chain polypeptide can be indirectly linked to an anchor via anintermediate association such as the high affinity interaction of theJun and Fos leucine zippers (jun/fos linkage) to covalently link apolypeptide chain to an anchor of a phage or host cell (Crameri, R. andSuter, M., 1993; Crameri, R. and Blaser. K., 1996).

[0094] In the particularly preferred embodiment, wherein the multi-chainpolypeptide is an Ig Fab fragment: the first polynucleotide comprises anAga2p signal sequence in frame with a segment that encodes an Aga2panchor, and in frame with a segment that encodes the V_(H) and C_(H)1domains of an Ig heavy chain; and the second polynucleotide comprises anAga2p signal sequence in frame with a segment that encodes an Ig lightchain.

[0095] Preferably, the multi-chain display vectors of the presentinvention provide cloning sites to facilitate transfer of thepolynucleotide sequences that encode the chains of a multi-chainpolypeptide. Such vector cloning sites comprise at least one restrictionendonuclease recognition site positioned to facilitate excision andinsertion, in reading frame, of polynucleotides segments. Any of therestriction sites known in the art can be utilized in the vectorconstruct of the present invention. Most commercially available vectorsalready contain multiple cloning site (MCS) or polylinker regions. Inaddition, genetic engineering techniques useful to incorporate new andunique restriction sites into a vector are known and routinely practicedby persons of ordinary skill in the art. A cloning site can involve asfew as one restriction endonuclease recognition site to allow for theinsertion or excision of a single polynucleotide fragment. Moretypically, two or more restriction sites are employed to provide greatercontrol of, for example, insertion (e.g., direction of insert), andgreater flexibility of operation (e.g., the directed transfer of morethan one polynucleotide fragment). Multiple restriction sites can be thesame or different recognition sites.

[0096] The multi-chain eukaryotic display vector of the presentinvention preferably contains restriction sites positioned at the endsof the coding sequences for the chains of the multi-chain polypeptide.Restriction sites can be positioned at the extreme ends, 5′ and 3′ ofthe polynucleotide segment including all of the coding sequences for thechains of a multi-chain polypeptide (on a single vector); or, morepreferably, restriction sites can be positioned at the 5′ and 3′ ends ofeach polynucleotide segment encoding a chain of the multi-chainpolypeptide. Most preferably each of the restriction sites is unique inthe vector and different from the other restriction sites. Thisparticularly useful vector construct provides flexibility and controlfor the modular transfer of individual polynucleotide sequences encodinga chain of a multi-chain polypeptide.

[0097] In a particularly preferred vector construct, wherein themulti-chain polypeptide is a Fab, the first polynucleotide comprises anAga2p signal sequence in frame with a segment that encodes an Aga2panchor, and in frame with a segment that encodes the V_(H) and C_(H)1regions of an Ig heavy chain, wherein the Ig heavy chain region isbordered by unique restriction sites (e.g., SfiI and NotI); and thesecond polynucleotide comprises an Aga2p signal sequence in frame with asegment that encodes an Ig light chain, wherein the Ig light chainregion is bordered by unique restriction sites (e.g., ApaLI, and AscI).

[0098] In a preferred embodiment of the multi-chain eukaryotic displayvector, one or more of the chains of the multi-chain polypeptideexpressed by the vector in a host cell is linked to a molecular tag orreporter gene. Preferably, the linkage is a peptide bond that links apolypeptide tag to a chain of the multi-chain polypeptide. One or morechains of the multi-chain polypeptide can be tagged using identical,similar or different tags. Preferred tags include epitope tags (Munro,S. and Pelham, H., 1987). Preferred epitope tags include polyHis tags,HA tags, and myc tags, and preferably each chain is fused to a differenttag.

[0099] Building upon the particularly preferred vector constructexemplified herein, wherein the multi-chain polypeptide is a Fabfragment of an immunoglobulin, the first polynucleotide comprises anAga2p signal sequence in frame with a segment that encodes an Aga2panchor, in frame with a segment that encodes the V_(H) and C_(H)1regions of an Ig heavy chain, and in frame with a segment that encodes amyc tag, wherein the Ig heavy chain region is bordered by uniquerestriction sites (e.g., SfiI and NotI); and the second polynucleotidecomprises an Aga2p signal sequence in frame with a segment that encodesa HA tag, and in frame with a segment that encodes an Ig light chain,wherein the Ig light chain region is bordered by unique restrictionsites (e.g., ApaLI, and AscI).

[0100] Eukaryotic Cell Display of a Multi-Chain Polypeptide

[0101] Utilizing the vector described and taught herein, a process fordisplaying a biologically active multi-chain polypeptide on the surfaceof a eukaryotic host cell is demonstrated herein for the first time. Theprocess for displaying a multi-chain polypeptide on the surface of aeukaryotic host cell comprises introducing the vector (possibly as avector set) into a eukaryotic cell (i.e., a host cell), and culturingthe host cell under conditions suitable for expression, transport, andassociation of the chains of the multi-chain polypeptide with the hostcell surface such that the biological activity of the multi-chainpolypeptide is exhibited at the surface of the host cell.

[0102] The mode of introduction of the vector of the present inventioninto a host cell is not limiting to the present invention and includesany method for introducing genetic material into a cell known in theart. Such methods include but are not limited to methods known andreferred to in the art as transfection, transformation, electroporation,liposome mediated transfer, biolistic transfer, conjugation, cellularfusion, and nuclear microinjection. Transformation techniques known inthe art are the preferred methods of genetic transfer.

[0103] Multi-Chain Polypeptide Display Host Cells (and Host Cell Pairs)

[0104] Vectors of the present invention are operable in a eukaryotichost cell to effect expression and to display a multi-chain polypeptideon the surface of the eukaryotic host cell. Optionally, particularly inthe case of dual display vectors, the vectors of the present inventionare operable in a prokaryotic host cell as well, to effect expression ina bacterial host cell and to display a multi-chain polypeptide on thesurface of a bacteriophage. The eukaryotic host cell can be anyeukaryotic cell, of any genotype, differentiated or undifferentiated,unicellular or multi-cellular, depending on the practitioner'sparticular interest and requirements. Particularly useful eukaryoticcells include mammalian cells, plant cells, fungus cells, and protistcells. Preferably, the host cell is an undifferentiated, unicellular,haploid or diploid cellular organism. Fungi are preferred host cells,particularly species of the phylum Ascomycota (sac fungi), because oftheir ease and diversity of culture conditions, the variety ofbiochemical and cellular mutants available, their short generation time,and their life cycle (see below). Preferred fungal host cells includethose of the genera Neurospora and the various yeasts, such asSaccharomyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces,Yarrowia, Debaryomyces, and Candida. Most preferred species isSaccharomyces cerevisiae (baker's yeast), perhaps the most well known,characterized, and utilized eukaryotic cell system in molecular biologyresearch.

[0105] In particular embodiments, the eukaryotic host cells are suitablefor cell fusion (see below). For example, yeast cells of opposite matingtype can be “mated” to produce fused diploid cells. In addition, yeastprotoplasts or spheroplasts suitable for cell fusion are also suitableeukaryotic host cells for the purposes of the invention. Alternatively,cells grown in culture (e.g., mammalian cells, insect cells, etc.), canbe fused by methods known in the art (e.g., using Sendai virus orelectric current).

[0106] Phage Display-Eukaryotic Display Transfer System

[0107] The technical advancement of the present invention to displaycomplex multi-chain polypeptides on the surface of a eukaryotic hostcell can be coupled with the power of phage display technology. Forexample, by employing a phage display-eukaryotic display transfer systemas described herein, practitioners can, for the first time, combine theimmense diversity provided by phage display libraries and phage displaytechnology with the cellular expression, processing, assembly, anddisplay provided by the aforementioned multi-chain eukaryotic displaytechnology. The transfer of nucleic acid sequence information between aphage display vector and the eukaryotic vector of the present inventioncan be achieved by a variety of genetic transfer methods known in theart (e.g., genetic engineering technology such as recombinant DNAtechnology). Preferred modes of transfer include techniques ofrestriction digestion, PCR amplification, or homologous recombination.

[0108] In one embodiment, a eukaryotic/prokaryotic multi-chain displayshuttle vector as described and taught herein is employed. The geneticcontrol elements of the dual display vector of the present inventionprovide, within a eukaryotic host cell, for the expression, processing,assembly, and display of a biologically active multi-chain polypeptideon the surface of the eukaryotic host cell transformed with the dualdisplay vector, as well as provide, within a prokaryotic host cell, forthe expression, processing, assembly, and display of a biologicallyactive multi-chain polypeptide on the surface of a bacteriophageinfected in the prokaryotic host cell.

[0109] In another embodiment, the phage display-eukaryotic displaytransfer system is performed by inserting chain-encoding polynucleotidesegments excised from a conventional phage display vector (i.e., abacteriophage engineered to display an exogenous polypeptide on thesurface of the phage particle) known in the art, into the multi-chaineukaryotic display vector of the present invention, thereby enablingexpression of the chain-encoding segments, and eukaryotic processing,assembly, and display of a biologically active multi-chain polypeptideon the surface of a eukaryotic host cell transformed with the eukaryoticdisplay vector. As described above, transfer of the polynucleotidesequences from a phage display vector to a multi-chain eukaryoticdisplay vector can be achieved by any genetic engineering techniqueknown in the art. Two particularly preferred methods include a singleexcision/insertion transfer method and a multiple (or modular)excision/insertion transfer method.

[0110] In a single excision/insertion transfer process, thepolynucleotide segments that encode the chains of a multi-chainpolypeptide are excised (e.g., via restriction digestion) from the phagedisplay vector as a single, unitary nucleic acid, and subsequentlyinserted into the multi-chain display vector. Once inserted into theeukaryotic display vector, unwanted prokaryotic genetic control elements(if any) positioned between the chain encoding polynucleotides arereplaced with eukaryotic genetic control elements. This process isdiagramed for an Ig Fab multi-chain polypeptide, transferred from aphage display vector to a particularly preferred multi-chain yeastdisplay vector of the present invention in FIG. 1.

[0111] Alternatively, polynucleotide segments encoding chains of amulti-chain polypeptide are excised from the phage display vectorindividually, and subsequently inserted into the multi-chain displayvector in a separate and independent manner. This approach providesgreater control and flexibility over the transfer of individual chainsof a multi-chain polypeptide separately or en masse. Indeed, dependingupon the practitioner's interests and needs, only select chains of themulti-chain polypeptide need to be transferred. This process isdiagramed for an Ig Fab multi-chain polypeptide in FIG. 2.

[0112] Practitioners skilled in the art will appreciate that the phagedisplay-eukaryotic display transfer system described and taught hereinis equally functional whether transferring sequence information from aphage display vector to a multi-chain eukaryotic display vector, or froma multi-chain eukaryotic display vector to a phage display vector; i.e.,the phage display-eukaryotic display transfer system of the presentinvention is effectively bi-directional. A particularly preferred phagedisplay library for use in the phage display-eukaryotic display transfersystem according to the invention is a large human Fab fragment library(de Haard, H. et al., 1999).

[0113] Multi-Chain Eukaryotic Display Libraries and Screening ProtocolsThereof

[0114] Multi-chain eukaryotic display vectors of the present invention,and host cells transformed with these vectors such that a biologicallyactive multi-chain polypeptide in displayed on the host cell surface,are useful for the production of display libraries. Such displaylibraries are, in turn, useful to screen for a variety of biologicalactivities of interest to the practitioner; e.g., to screen against anyof a variety of target molecules to identify binding polypeptidesspecific for that target.

[0115] Several methods exist for expressing a variable array ofmolecules on the surface of a host cell or phage. Phage displaylibraries, and the screening of the same, represent a powerful researchand development tool. Methods for producing and screening phage displaylibraries are well known and used in the art (Hoogenboom, H. et al.,1997; Kay et al., 1996; Ladner, R et al., 1993).

[0116] The multi-chain eukaryotic display vectors of the presentinvention can be used to generate novel peptide libraries de novo,similar to known phage display libraries. However, the vectors describedherein provide allow for more efficient expression of properly folded,assembled, glycosylated, and displayed multi-chain polypeptides, as canonly be achieved in a eukaryotic system. These multi-chain eukaryoticdisplay libraries can then be used in screening assays. Persons ofordinary skill in the art will appreciate and easily adapt displaylibrary screening protocols known in the art (e.g., phage display screenassays) to the multi-chain eukaryotic display libraries of the presentinvention.

[0117] In addition to generating novel multi-chain eukaryotic displaylibraries de novo, the present invention further enables thepractitioner to transfer existing phage display libraries to themulti-chain eukaryotic display system disclosed and taught herein. Inparticular, the phage display-eukaryotic display transfer system allowsa phage display library to be constructed for the display of a verylarge repertoire of multi-chain polypeptides; for example Fabs, whichhave light chain and heavy chain components. The phage display library,which can have a diversity of >1×10⁸ (preferably >1×10⁹, morepreferably >1×1010) different multi-chain polypeptides in a library, canundergo an initial screen, producing a subpopulation of less than about1×10⁷ (preferably between 1×10⁵ to 1×10⁶) phage display isolates. Thepolynucleotides encoding the chains of the multi-chain polypeptideisolates can then be “batch transferred” to a multi-chain eukaryoticdisplay vector of the present invention for transformation into aeukaryotic host. The multi-chain polypeptides displayed on eukaryotichost cells can be further screened and manipulated, taking advantage ofthe culture conditions and expression qualities of the eukaryotic hostsystem as discussed earlier (e.g., protein folding, proper associationof separate chains in the multi-chain protein, glycosylation, secretion,and post-translational modifications such as phosphtidyl inositollinkages to the cell membrane). In addition, once inserted into themulti-chain eukaryotic display vector, the multi-chain polypeptidelibrary (or pre-selected isolates therefrom) can be further diversified(e.g., polypeptide chain recombination, re-shuffling, or re-mixing) foradditional rounds of screening.

[0118] In a particularly preferred embodiment, a M13 phage expressionvector is provided having:

[0119] an Ig light chain cloning site defined by an ApaLI restrictionsite and an AscI restriction site, and which is oriented 3′ to a signalsequence (e.g., a pIII signal sequence) and under the transcriptionalcontrol of a LacZ promoter; and

[0120] an Ig heavy chain fragment cloning site defined by a SfiIrestriction site and a NotI restriction site, and which is oriented 3′to a signal sequence (e.g., a pIII signal sequence), under thetranscriptional control of a LacZ promoter, and 5′ to a sequenceencoding mature pIII or an anchoring portion of pIII (stump).

[0121] The multi-chain eukaryotic display vector in this preferredembodiment is a yeast vector having:

[0122] an Ig light chain cloning site defined by an ApaLI restrictionsite and an AscI restriction site, and which is oriented 3′ to an Aga2psecretion signal and under the transcriptional control of a GAL promoter(preferably GAL1 or GAL1-10); and

[0123] an Ig heavy chain fragment cloning site defined by a SfiIrestriction site and a NotI restriction site, and which is oriented 3′to an Aga2p secretion signal, under the transcriptional control of a GALpromoter (preferably GAL1 or GAL1-10), and 3′ to a sequence encodingmature Aga2p.

[0124] The yeast expression vector is used to transform a yeast hostcell for expression of antibodies or Fab fragments displayed on theyeast cell surface. Light and heavy chain coding sequences are excisedindividually (by ApaLI/AscI digestion and SfiI/NotI digestionrespectively), or together (by ApaLI/NotI digestion) from the phagedisplay vector, and inserted into the multi-chain yeast display vectorby batch transfer, yielding a multiplicity of LC/HC chain pairings forexpression and display in yeast. A particularly preferred yeast displayvector for yeast display of Fabs is pTQ3 (described below). Aparticularly preferred phage display is a large human Fab fragmentlibrary (de Haard, H. et al., 1999).

[0125] It will be appreciated by one skilled in the art that the abovemethods are useful for identifying and isolating multi-chainpolypeptides possessing a variety of detectable characteristics (e.g.,catalytic activity, peptide interactions, thermal stability, desirableexpression levels) or any other improvement that is selectable viasurface expression of a displayed multi-chain polypeptide.

[0126] It will be further appreciated that the present invention can beused for the production of antibodies or antibody fragments useful forimmunopurification, immunoassays, cytochemical labeling and targetingmethods, and methods of diagnosis or therapy. For example, the antibodyor fragment can bind to a therapeutically active protein such asinterferon or a blood clotting factor such as, for example, Factor VIII,and can therefore be used to produce an affinity chromatography mediumfor use in the immunopurification or assay of the protein.

[0127] Multi-Chain Polypeptide Display as a Product of Cellular Fusion

[0128] The basic life cycle of eukaryotic cells involves an alternationbetween diploid (two copies of an organism's chromosomes or genome percell) and haploid (one copy of an organism's chromosomes or genome percell) states. The alternation between these two states is achieved bythe fusion of two haploid cells (typically, although not necessarily,the fertilization of opposite mating types) to form a single diploidcell, and meiotic division of a diploid cell to form multiple haploid(daughter) cells. Biologists appreciate that this basic life cycle(i.e., the alternation of haploid and diploid generations) provides animportant natural mechanism for the biological recombination geneticinformation (i.e., sexual reproduction).

[0129] In most animals, the diploid state is the dominant stage of thelife cycle, generated by the fusion of two haploid cells (commonlyreferred to as gametes) of opposite mating type; a sperm and an egg.Meiotic cell division of diploid cells (gametogenesis) produces thehaploid cell state for sexual reproduction.

[0130] The life cycle pattern of the plant kingdom provides a moregeneral alternation of generation wherein the haploid and diploid statecan exist as more distinct generations, depending on the particularplant species. In “lower” (i.e., more primitive) plants, the generationof the haploid cell (the “gametophyte”) predominates (e.g., mosses,liverworts, and homworts); whereas in “higher” (i.e., more advanced)plants, the generation of the diploid cell (the “sporophyte”)predominates (e.g., ferns, conifers, and flowering plants).

[0131] For many fungi and protists, the haploid stage of the life cyclepredominates. Fertilization produces a diploid stage, which often almostimmediately (depending upon environmental conditions) undergoes meiosisto form haploid cells. Importantly, and regardless of which genera oforganism is being discussed or which stage dominates the organism's lifecycle, the natural recombination and re-mixing of genetic material thatresults from meiosis of diploid cells to produce haploid cells, and thecellular fusion of separate haploid cells to produce diploid cell (of anew genetic admixture) is a powerful process that can be utilized inbiological research. Described and taught herein for the first time,this powerful mechanism is utilized in combinatorial protein researchfor the generation of unique multi-chain peptide display libraries.

[0132] In a further aspect of the present invention, the mode forintroducing eukaryotic multi-chain display vectors into a host cellincludes the fusion of two eukaryotic cells, preferably haploid, eachexpressing at least one of the chains of the multi-chain polypeptide,such that the biological activity of the multi-chain polypeptide isexhibited at the surface of the resulting host cell, preferably diploid.For example, each of the two haploid cells can contain one of thevectors of a vector set (as described herein), such that once combined(e.g., via cellular fusion of host cells) and co-expressed in theresulting diploid host cell, the biological activity of the multi-chainpolypeptide is exhibited at the surface of the host cell. Such methodscan be used to prepare novel multi-chain polypeptide libraries asdescribed above (for example, antibody or Fab display libraries,including diploid host cells displaying multi-chain polypeptides havinga greater diversity than the source repertoire).

[0133] Alternatively, populations of a matched vector set can beconstructed such that one eukaryotic expression vector populationexpresses multiple (e.g., a repertoire or library) forms of an Ig Fablight chain (comprising V_(L) and C_(L) domains) and a second eukaryoticexpression vector population expresses multiple forms of an Ig Fab heavychain (comprising V_(H) and C_(H)1 domains) fused to a yeast anchorprotein (e.g., Aga2p). Each of the vector populations are used totransform haploid yeast cells of opposite mating type; one vectorconstruct in one mating type, the second vector construct in theopposite mating type. The two haploid yeast populations are co-culturedunder conditions sufficient to induce yeast mating (i.e. cellularfusion) of the two mating types. The resulting diploid yeast host cellsof the population possess both vector constructs and expresses anddisplays the fully formed and assembled Ig Fab.

[0134] Although, as discussed above, any eukaryotic cell capable of cellfusion can be used in the present invention. Cell fusion can occursexually by mating, or artificially, e.g., in tissue culture or otherartificial conditions. In the case of sexual cell fusion, any eukaryoticcell is suitable as long as it is capable of existing (no matter howbriefly) in both a haploid and a diploid state. For artificial cellfusion, cells are not limited by ploidy as they would be in the case ofsexual fusion. For example, diploid mammalian cells maintained in tissueculture can be induced to fuse, thereby resulting in a tetraploid hostcell. For the present invention, the actual ploidy of the host cells tobe fused does not pose a limitation so long as the cells can be fused.The important features of the cells are that one cell partner contains avector or vector set comprising a particular chain of a multi-chainpolypeptide and a specific selectable marker, and the partner host cellcontains a vector or a vector set comprising a second chain of amulti-chain polypeptide and a selectable marker. When the cells arefused, therefore, the resultant fused cell contains vectors encoding twoor more chains of a multi-chain polypeptide in a cell that is readilyidentified by the selectable markers.

[0135] Fungi, especially sac fungi (ascomycetes; e.g., Neurospora andyeasts), are particularly preferred eukaryotic host cells. Sac fungi areso named because they produce the haploid spore products of meiosis inmicroscopic sacs, which render them easily collected, segregated,analyzed, and manipulated (Neurospora are particularly noted because thesize and shape of their ascus maintains the order of the haploid cellproducts of meiosis). Also, these fungi, especially S. cerevisiae, existstably in both haploid and diploid form, either of which are easilyinduced and maintained (e.g., the yeast haploid state is typicallyinduced and maintained under some form of nutritional stress, i.e.,starvation). Finally, in many fungi (again especially preferred yeast)haploid cells exist as two sexes (the α and α mating types), from whichonly opposite mating types fuse (mate) to form the diploid state. Underconditions manipulable in the lab by one of skill in the art, an α cellwill fuse to an a cell, thereby creating a fused diploid cell.

[0136] As noted above, artificial methods of fusing cells are known inthe art.

[0137] Therefore the present invention is suitable for eukaryotic cellssuch as, for example, mammalian, insect or plant cells that are grown inculture. Additionally, yeast protoplasts or spheroplasts can bemanipulated to undergo cell fusion even if they are of the same matingtype. Such artificial methods for cell fusion are known in the art andwould be suitable for the purposes of the present invention.

[0138] Finally, practitioners skilled in the art will recognize andappreciate that the products and methods described and demonstratedherein are not limited by a eukaryotic host cell of a particular ploidy.Indeed, other polyploid organisms (e.g., rarer triploid and tetraploidforms) can be used especially as hosts for matched vector setsexpressing higher order multi-chain polypeptides (e.g., three-chain andfour-chain polypeptides respectively).

[0139] Multi-chain Polypeptide Screening Using a Eukaryotic CellularFusion

[0140] Multi-chain polypeptides libraries displayed on eukaryotic hostcells can be screened and manipulated similar to procedures andtechniques known in the art, e.g., phage display library screening, butalso allow the practitioner to take advantage of culture conditions andexpression qualities of a eukaryotic host system. As discussed above,eukaryotic display screening can be prefaced with an initial round ofphage display screening before transferring the display library from thephage display vector to a multi-chain eukaryotic display vector. Onceinserted into the multi-chain eukaryotic display vector, the multi-chainpolypeptide library (or pre-selected isolates) can be subjected to oneor more additional rounds of screening under the eukaryotic displaysystem.

[0141] As a further embodiment of the screening methods of the presentinvention, and unique to the methods of the present invention,multi-chain eukaryotic display libraries can undergo further (biased orunbiased) diversification subsequent to any screen assay utilizing thealternation of generations characteristic of eukaryotic systems asdiscussed above. Populations of diploid eukaryotic host cells containinga multi-chain eukaryotic display vector, wherein different chains of themulti-chain are expressed from different vectors (e.g., where thediploid host cell is the product of haploid mating or cell fusions asdescribed above), can be induced to undergo meiosis (e.g., sporulationin yeast). The haploid yeast cells (spores) can be segregated and/orselected depending on screening conditions in order to isolate differenteukaryotic expression vectors with a preferred property of interest inseparate haploid daughter cells. The daughter cells can then beoptionally:

[0142] mutagenized (variegated) in vitro (e.g., isolated DNAmanipulation) or in vivo (e.g., UV light) to provide a multiplicity ofhomologs of the pre-selected chains. When these homologous chains areco-expressed and displayed, homologous multi-chain polypeptides havinggreater affinities for the same target molecule can be selected; or

[0143] fused back together with other daughter host cells, thusrecombining individual pre-selected chains of the multi-chainpolypeptide isolates among themselves; or

[0144] fused with the initial multi-chain library host cell population,thus recombining pre-selected chains of the multi-chain polypeptideisolates with the original source of multi-chain variation; or

[0145] fused with a new multi-chain library host cell population; thuscombining the pre-selected chains of the multi-chain polypeptideisolates with a new source of multi-chain variability; or

[0146] any combination of any of the above steps as appropriate.

[0147] Once this recombination, re-shuffling, or re-mixing ofpre-selected chains of a multi-chain polypeptide screen among themselvesor with another source of multi-chain diversity is complete, the newadmixture library population can undergo further rounds of new or repeatscreening.

[0148] The present invention incorporates by reference in their entiretytechniques well known in the field of molecular biology. Thesetechniques include, but are not limited to, techniques described in thefollowing publications:

[0149] Ausubel, F. et al., eds., Short Protocols In Molecular Biology(4th Ed. 1999) John Wiley & Sons, NY, N.Y. (ISBN 0-471-32938-X).

[0150] Fink and Guthrie, eds., Guide to Yeast Genetics and MolecularBiology (1991) Academic Press, Boston, Mass. (ISBN 0-12-182095-5).

[0151] Kay et al., Phage Display of Peptides and Proteins: A LaboratoryManual (1996) Academic Press, San Diego, Calif.

[0152] Kabat, E. et al., Sequences of Proteins of Immunological Interest(5th Ed. 1991) U.S. Dept. of Health and Human Services, Bethesda, Md.

[0153] Lu and Weiner, eds., Cloning and Expression Vectors for GeneFunction Analysis (2001) BioTechniques Press. Westborough, Mass. (ISBN1-881299-21-X).

[0154] Old, R. and Primrose, S., Principles of Gene Manipulation: AnIntroduction To Genetic Engineering (3d Ed. 1985) Blackwell ScientificPublications, Boston, Mass. Studies in Microbiology; V.2:409 (ISBN0-632-01318-4).

[0155] Sambrook, J. et al., eds., Molecular Cloning: A Laboratory Manual(2d Ed. 1989) Cold Spring Harbor Laboratory Press, NY, N.Y. Vols. 1-3(ISBN 0-87969-309-6).

[0156] Winnacker, E., From Genes To Clones: Introduction To GeneTechnology (1987) VCH Publishers, NY, N.Y. (translated by HorstIbelgaufts). (ISBN 0-89573-614-4).

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[0171] Phizicky, E. and Fields, S., 1995. Microbiol. Rev., 59:94-123.

[0172] Pu, W. and Struhl, K., 1993. Nucleic Acids Res., 21:4348-55.

[0173] Walhout, A. et al., 2000. Methods Enzymol., 328:575-593.

[0174] Wittrup et al., WO 99/36569.

[0175] This invention is illustrated further by the following examples,which are not to be construed as limiting in any way.

EXAMPLES Example 1 Construction of a Multi-Chain Eukaryotic DisplayVector: pTQ3

[0176] The materials and techniques described above and incorporated byreference were used to construct a multi-chain eukaryotic displayvector; specifically a yeast display vector effective in a host yeastcell transformed with the vector. The vector is useful for expressing,transporting, assembling and displaying a biologically activemulti-chain polypeptide (e.g., an Ig Fab) on the surface of the hostyeast cell.

[0177] In this example, a commercially available vector, pYD 1(InVitrogen, Carlsbad, Calif.), a 5.0 kb expression vector designed forexpression, secretion, and display of a single chain protein on thesurface of S. cerevisiae cells, was used as the starting eukaryoticexpression vector template. pYD1 includes: an aga2 gene encoding one ofthe subunits of the α-agglutinin receptor; a GAL1 promoter for regulatedexpression of an Aga2/polypeptide fusion; an HA epitope tag fordetection of the displayed protein; a polyhistidine (6×His) tag forpurification on metal chelating resin; a CEN6/ARS4 for stable, episomalreplication in yeast; and a TrpI gene for S. cerevisiae transformantselection, an ampicillin resistance gene (ampR) and the pMB 1 origin forselection and replication in E. coli.

[0178] The pYD1 plasmid was modified for expression of an Ig light chainand a heavy chain fragment from two tandem galactose induciblepromoters, for the display of an intact Fab antibody fragment. One GAL1promoter directs expression of the light chain and the other GAL1promoter directs expression of the heavy chain fragment fused to theC-terminus of the Aga2p yeast anchor protein.

[0179] In order to effectively transfer the chains of a multi-chainpolypeptide into the display vector, unique restriction sites weregenerated as part of the vector construct. The restriction endonucleaserecognition sequences (i.e., restriction sites) chosen for this vectorconstruct included ApaLI, AscI, SfiI, and NotI as the unique cloningsites for the chains of a two-chain polypeptide (in this case an IgFab), and NheI to facilitate phage display-eukaryotic display transferswith existing phage display libraries.

[0180] Several vector sequence modifications were made to ensureeffective use of ApaLI as a unique restriction site. The ApaLI siteslocated on the pYD1 plasmid (as supplied by Invitrogen) starting atpositions 1393, 3047, and 4293 were removed by site-directed mutagenesis(using QUICKCHANGE, Stratagene, La Jolla, Calif.) as indicated below:pYD1 position ApaLI nucleotide change 1393 GTGCAC to GTGCAG 3047 GTGCACto GTGCTC 4293 GTGCAC to GAGCAC

[0181] The ApaLI site beginning at position 3047 lies within the ampR,requiring a silent mutation so as not to change the amino acid codingsequence of this gene.

[0182] In order to render the multi-chain yeast display vector constructcompatible with other pre-existing phage display vectors known in theart (Dyax Corp., Cambridge, Mass.), a unique restriction site wasintroduced into the pYD1 vector aga2p signal sequence without alteringthe coding sequence, using PCR site directed mutagenesis techniquesknown in the art. Specifically, a NheI site was created across theterminal serine codon of the aga2p signal sequence by replacing codonTCA with codon AGC.

[0183] The vector thus modified having a unique ApaLI site immediately3′ to the pre-existing GAL1 promoter-aga2p signal sequence-HA tagsegments, followed by a AscI site, and a NheI site incorporated in theaga2p signal sequence was designated pTQ2.

[0184] Assembly PCR techniques known in the art were used to construct apolylinker compatible with existing phage display libraries forexcision/insertion of structural genes for the light chain component ofa Fab into the multi-chain yeast display vector. The resultingintermediate multi-chain eukaryotic display vector segment spanning theapa2p signal sequence through the designed polylinker site is as follows(*indicates stop codons):                                                     NheI ATG CAG TTACTT CGC TGT TTT TCA ATA TTT TCT GTT ATT GCT AGC GTT (SEQ ID NO:1) M   Q   L   L   R   C   F   S   I   F   S   V   I   A   S   V (SEQ IDNO:2)                                 Aga2p signal sequence                                             ApaLI TTA GCA TAC CCA TACGAC GTT CCA GAC TAC GCT AGT GCA CAG GAT L   A   Y   P   Y   D   V   P   D   Y   A   S   A   Q   D                    HA epitope tag                  AscI      BamHI                    PstI TTC GTG CAATGC GGC GCG CCA GGA TCC GCC TGA ATG GTC TGC AGA F   V   Q   C   G   A   P   G   S   A   *   M   V   C   R               EcoRI                       PacI CCG TAC CGACCG AAT TCG AGT TAC CTG AGG TTA ATT AAC ACT GTT P   Y   R   P   N   S   S   Y   L   R   L   I   N   T   V        PmeIATC GTT TAA ACG TTC AGG TGC AA  I   V   *   T   F   R   C

[0185] A MATα transcriptional terminator sequence was amplified by PCRfrom the pYD 1plasmid, and BamHI and PstI restriction sites wereappended to facilitate cloning into plasmid pTQ2 above. The MATαterminator was then digested with BamHI and PstI and inserted into theBamHI/PstI site on plasmid pTQ2.

[0186] A DNA construct including (5′-3′); the GAL1 promoter, the aga2psignal sequence, the Aga2p protein coding sequence, and a glycine/serinelinker, was amplified from plasmid pYD1. A DNA linker segment containingSfiI and NotI restriction sites and a segment coding for a myc tag wereadded at the 3′ end of the amplified pYD1 segment. The myc tag wasincluded to allow detection of the anchored chain (of the multi-chainpolypeptide) on the yeast cell surface. The linker-myc segment sequencesis as follows: GGA GGC GGA GGT TCT GGG GGC GGA GGA TCT GGT GGC GGA GGTTCT (SEQ ID NO:3) G   G   G   G   S   G   G   G   G   S   G   G   G   G   S (SEQ ID NO:4)       SfiI                       NotI GCG GCC CAG CCG GCC AGT CCT GATGCG GCC GCA GAA CAA AAA CTC G   G   Q   P   G   S   P   D   A   A   A   E   Q   K   L                     PacI ATC TCA GAA GAG GAT CTG AAT TTA ATTAA I   S   E   E   D   L   N

[0187] This linker-myc segment was inserted into an EcoRI and PacIdigested pTQ2. The resulting plasmid, with unique cloning sites forinsertion/excision of the chains of a multi-chain polypeptide(specifically light chain and heavy chain fragments of a Fab), wasdesignated pTQ3 (FIG. 3). Plasmid pTQ3 is a 5810 bp multi-chain yeastdisplay plasmid comprising, in pertinent part, the following vectorsequence: <-----------------------Aga2p signal sequence---------- 435ATG CAG TTA CTT CGC TGT TTT TCA ATA TTT TCT GTT ATT GCT (SEQ ID NO:6) M   Q   L   L   R   C   F   S   I   F   S   V   I   A--------------> <-------------HA tag--------------> AGC GTT TTA GCA TACCCA TAC GAC GTT CCA GAC TAC GCT S   V   L   A   Y   P   Y   D   V   P   D   Y   A   ApaLI                          AscI       BamHI AGT GCA CAG GAT TTCGTG CAA TGC GGC GCG CCA GGA TCC S   A   Q   D   F   V   Q   C   G   A   P   G   S ATG TAA  M<---------------------Mat α terminator---------------------> 661CAAAATCGACTTTGTTCCCACTGTACTTTTAGCTCGTACAAAATACAATATACTTTTCAT 721TTCTCCGTAAACAACATGTTTTCCCATGTAATATCCTTTTCTATTTTTCGTTCCGTTACC 781AACTTTACACATACTTTATATAGCTATTCACTTCTATACACTAAAAAACTAAGACAATTT 841TAATTTTGCTGCCTGCCATATTTCAATTTGTTATAAATTCCTATAATTTATCCTATTAGT                          EcoRI 901AGCTAAAAAAAGATGAATGTGAATCGAATCCTAAGAGAATTCACGGATTAGAAGCCGCCG<----------------------GAL1 promotor-----------------------> 961AGCGGGTGACAGCCCTCCGAAGGAAGACTCTCCTCCGTGCGTCCTCGTCTTCACCGGTCG 1021CGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAA 1081TACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTC 1141AAATGAACGAATCAAATTAACAACCATAGGATGATAATGCGATTAGTTTTTTAGCCTTAT 1201TTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGC 1261AAAAACTGCATTAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTT 1321ATTCAAATGTAATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTC 1381AAGGAGAAAAAACCCGGATCGGACTACTAGCAGCTGTAATACGACTCACTATAGGGAATA 1441TTAAGCTAATTCTACTTCATACATTTTCAATTAAG <-------------------------Aga2psignal sequence------------------------ 1476 ATG CAG TTA CTT CGC TGT TTTTCA ATA TTT TCT GTT ATT GCT TCA GTT TTA GCA M   Q   L   L   R   C   F   S   I   F   S   V   I   A   S   V   L   A--------------> <----------------Mature Aga2 protein-------------------1530 CAG GAA CTG ACA ACT ATA TGC GAG CAA ATC CCC TCA CCA ACT TTA GAA TCGACG Q   E   L   T   T   I   C   E   Q   I   P   S   P   T   L   E   S   T-----------------------------------------------------------------------1584 CCG TAC TCT TTG TCA ACG ACT ACT ATT TTG GCC AAC GGG AAG GCA ATG CAAGGA P   Y   S   L   S   T   T   T   I   L   A   N   G   K   A   M   Q   G-----------------------------------------------------------------------1638 GTT TTT GAA TAT TAC AAA TCA GTA ACG TTT GTC AGT AAT TGC GGT TCT CACCCC V   F   E   Y   Y   K   S   V   T   F   V   S   N   C   G   S   H   P----------------------------------------------------------> 1692 TCA ACGACT AGC AAA GGC AGC CCC ATA AAC ACA CAG TAT GTT TTT S   T   T   S   K   G   S   P   I   N   T   Q   Y   V   F<----------Glycine-Serine linker--------------------------> 1736 GGA GGCGGA GGT TCT GGG GGC GGA GGA TCT GGT GGC GGA GGT TCT G   G   G   G   S   G   G   G   G   S   G   G   G   G   S         SfiI                       NotI    <----------myc tag-------->1782 GCG GCC CAG CCG GCC AGT CCT GAT GCG GCC GCA GAA CAA AAA CTC ATC TCAGAA (SEQ ID NO:5) A   A   Q   P   A   S   P   D   A   A   A   E   Q   K   L   I   S   E(SEQ ID NO:7) -------------->   PacI                    PmeI 1836 GAGGAT CTG AAT TTA ATT AAC ACT GTT ATC GTT TAAAC E   D   L   N   L   I   N   T   V   I   V

[0188] Later modifications were made to the above vector by inserting a6×His tag for purification of soluble Fab antibodies and byrepositioning the stop codon (TAA) at the end of the myc tag, before thePacI site, to eliminate superfluous amino acids. Other modificationshave included the removal of an endogenous XbaI restriction site withinthe Trp selective marker by site directed mutagenesis. This was done tofacilitate cloning and manipulation of lead antibodies from the CJlibrary set (Dyax Corporation, Cambridge, Mass.).

Example 2 Phage Display-Eukaryotic Transfer and Eukaryotic Host CellExpression of Multi-Chain Fab Polypeptides Specific for Streptavidin,Mucin-1 and Cytotoxic T-Lymphocyte Associated Antigen 4

[0189] Different phage display Fabs were transferred from the phagedisplay vector to a multi-chain eukaryotic display vector to demonstrateof the utility of the phage display-eukaryotic display transfer system,and the ability of the multi-chain eukaryotic vector of the presentinvention to express a multi-chain polypeptide. The vector was theninserted into a eukaryotic host cell, and the transformed host cellgrown under conditions suitable for expression of the Fabs.

[0190] Anti-streptavidin Fab antibodies, F2, A12, and 4C8 were eachcloned from a large naive human Fab library (de Haard, H. et al., 1999)into the multi-chain yeast display vector pTQ3 constructed in Example 1as a paired light chain (V_(L)C_(L)) and heavy chain (V_(H)C_(H)1).Additionally, an anti-mucin Fab antibody, PH1, was cloned from the sameFab library into the multi-chain yeast display vector pTQ3 constructedin Example 1 as a paired light chain (V_(L)C_(L)) and heavy chain(V_(H)C_(H)1). Additionally, four antibodies, E7, E8, A9, A11, specificfor cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) were clonedfrom the same Fab library into the multi-chain yeast display vector pTQ3constructed in Example 1 as a paired light chain (V_(L)C_(L)) and heavychain (V_(H)C_(H)1).

[0191] The chains of the Fabs were cloned into the multi-chain yeastdisplay vector using the single excision/insertion transfer processdescribed earlier and illustrated in FIG. 1. The LC-HC polynucleotidefrom the Fab library was inserted as a single ApaLI/NotI fragment. Theunwanted prokaryotic genetic control elements intervening the codingregions of the LC and HC fragment and defined by the AscI/SfiIrestriction fragment from the Fab library was replaced with theAscI/SfiI fragment derived from pTQ3.

[0192] The resulting plasmids, designated pTQ3-F2, pTQ3-A12, pTQ3-4C8,pTQ3-PH1, pTQ3-E7, pTQ3-E8, pTQ3-A9 and pTQ3-A11, were separatelytransformed into S. cerevisiae strain EBY 100 (InVitrogen, Carlsbad,Calif.) following the method of Gietz, D. et al., 1992. EBY100 was alsotransformed pTQ3 containing no multi-chain insert as a control.Transformant selection was performed selecting for the vector tryptophanauxotrophic marker (synthetic defined medium minus tryptophan, 2% (w/v)glucose, 2% agar (SDCAA+GA)).

[0193] Successful transformants (correspondingly designated “EBY100pTQ3-F2”, “EBY100 pTQ3-A12”, “EBY100 pTQ3-4C8”, “EBY100 pTQ3-PH1”,“EBY100 pTQ3-E7”, “EBY100 pTQ3-E8”, “EBY100 pTQ3-A9”, “EBYpTQ3-A11”, andthe control “EBY100 pTQ3”) were grown overnight at 30° C. with shakingin 10 mL SDCAA+G. Two samples of cells were immediately removed when theOD₆₀₀ reached 1.0 (e.g., 2 mL of a culture of OD₆₀₀ of 1.0) for proteinlysate preparation as the time equals zero induction point (T₀). Thefollowing day, cultures were centrifuged and the pelletted yeast cellswere resuspended in 10 mL SDCAA, 2% (w/v) galactose to an OD₆₀₀ of 1.Cells were grown at 20° C. to induce vector expression of the light andheavy chains for 48 hours. Cultured cells were then centrifuged andwashed twice in 1 mL sterile water, and transferred to an eppendorf tubefor centrifugation.

[0194] Cell pellets were resuspended in 250 mL of SDS-PAGE buffer plusdithiothreitol (DTT). 425-600 micron glass beads (Sigma, St. Louis, Mo.)were added to just below the meniscus, and the suspension was vortexed 4times for 1 minute. The suspension was kept on ice between vortexing.The supernatant was transferred to a fresh tube and heated to 100° C.for 5 minutes.

[0195] Protein samples were separated on a SDS-PAGE gel and transferredto a nitrocellulose membrane for western blotting. Detection of thelight chain polypeptide was performed using an anti-HA antibody (1μg/mL) (Dako, Carpinteria, Calif.). Detection of the heavy chain-Aga2pfusion polypeptide was performed using an anti-c-Myc antibody (1 μg/mL)in conjunction with a secondary rabbit anti-mouse HRP antibody (Dako,Carpinteria, Calif.). Immunodetection was by enhanced chemiluminescence(Amersham-Pharmacia, Piscataway, N.J.). The LC product of approximately30 kD and the HC-Aga2p fusion product of approximately 45 kD of thedisplayed Fabs (F2 and PHI; FIGS. 4A and 4B) were detected. Nodetectable LC or HC-Aga2p fusion product was detected prior to inductionwith galactose (see FIGS. 4A and 4B).

Example 3 Functional Surface Display of a Multi-Chain Polypeptide on aEukaryotic Host Cell

[0196] As a demonstration of the ability of the multi-chain eukaryoticvector of the present invention to express, assemble and properlydisplay a biologically active multi-chain polypeptide on the surface ofa eukaryotic host cell, a multi-chain eukaryotic display vector wasinserted into a eukaryotic host cell and the transformed host cell wasgrown under conditions suitable for expression and display of the Fab onthe surface of the host cell.

[0197] Yeast clones EBY100 pTQ3-F2, EBY100 pTQ3-PHI, EBY100 pTQ3-E7,EBY100 pTQ3-E8, EBY100 pTQ3-A9 and EBY100 pTQ3-A11 were prepared,cultured, and induced for antibody expression as described in Example 2above. Three 0.2 mL aliquots of yeast cells having an OD₆₀₀ of 1.0 wereremoved prior to induction with galactose, as the T₀ point.

[0198] After inducing expression with galactose (Example 2), threeadditional 0.2 mL aliquots of cells having an OD₆₀₀ of 1.0 were removed.Yeast samples were centrifuged and the cell pellet resuspended in PBScontaining 1 mg/mL BSA.

[0199] Two samples were again centrifuged and the cell pelletsresuspended in either 100 mL of anti c-Myc antibody (2.5 μg per sample),or 100 mL of anti-HA antibody (2.0 μg antibody per sample). The sampleswere then incubated for one hour at room temperature, and the cellspelleted and washed once with 0.5 mL of PBS/BSA. The samples were thenincubated with FITC-conjugated rabbit anti-mouse antibody (1:40dilution) for 1 hour in the dark.

[0200] Cell samples were labeled with streptavidin-FITC (1:20 dilution)in PBS/1% (w/v) BSA and incubated overnight in the dark at roomtemperature. All samples were centrifuged and the cell pellets washedonce with 0.5 mL PBS and then resuspended in 500 mL of PBS.

[0201] The presence of cell surface bound Fab-antigen binding wasdetected by flow cytometry. Cells prior to induction showed no displayof light chain, heavy chain or functional streptavidin binding Fabantibody. After induction of Fab expression, yeast cells could bedetected displaying LC, HC and also functional streptavidin binding Fabantibody by immunofluorescence (FIG. 4C), by FACS (FIGS. 5A-C) and yeastwhole cell ELISA (FIG. 6, see Example 7). Functional display of theanti-CTLA-4 Fab antibodies was also demonstrated (data not shown).

[0202] In the case of EBY100 pTQ3-F2 and EBY100 pTQ3-PH1 antigen bindingas detected by FACS could be competed with unlabeled soluble antigen.Competitive binding showed the absolute specificity of thecombinatorially assembled Fab antibody displayed on the yeast cellsurface (FIG. 5C).

Example 4 Preferential Enrichment of Fab-Displaying Yeast Cells:Detection by Magnetic Bead Selection

[0203] To demonstrate that yeast cells displaying an antigen-specificFab antibody can be enriched over an excess of non relevant yeast cells,model selection experiments were performed using an automated magneticbead selection device.

[0204] The Fab-displaying yeast cell EBY100 pTQ3-F2 (with a tryptophanauxotrophic selectable marker) were mixed with nonspecific yeast cellsat various ratios. The non specific yeast cells consisted of EBY100pUR3867 (Unilever Research, VLaardingen, Netherlands), and encoding ascFv antibody specific for mucin-1 (PHI), and carrying a leucineauxotrophic selectable marker. The ratio of Leu⁺/Trp⁺ cells before andafter selection was used to calculate the enrichment factor after 1round of selection.

[0205] Yeast clones were grown and antibody expression induced withgalactose as described in Example 2. The two yeast clones were mixed inthe ratio indicated above, and incubated for 1 hour with 100 μLstreptavidin paramagnetic beads (Dynal M280, Dynal Biotech, Oslo,Norway) in an end volume of 1 mL 2% a phosphate-buffered saline solution(e.g., 2% MARVEL-PBS or “MPBS”, Premier Brands Ltd., U.K.).

[0206] After incubation of the yeast-bead mixture, the cell-beadcomplexes were washed for 11 cycles in 2% MPBS by transferring thecomplexes from one well to the next well in an automated magnetic beadselection device. After the 2% MPBS washing, two more washing steps wereperformed with PBS. In the last well of the automated magnetic beadselection device, the cell-beads complexes were resuspended in 1 mL PBSand the titres determined by plating on SDCAA+G agar plates or withsynthetic defined medium containing 2% (w/v) glucose containing leucinedrop out media plus 2% agar (SD-Leu+G agar plates). For selection bymagnetic activated cell sorting (MACS) yeast cells were incubated forone hour at room temperature with 500 μL streptavidin microbeads(Miltenyl Biotec, Cologne, Germany) in 6 mL PBS+2 mM EDTA. The cell/beadmixture was loaded onto a pre-washed LS column (Miltenyi Biotec,Cologne, Germany) in the presence of a magnet, and the column was washedtwice with PBS+2 mM EDTA. After the magnet was removed the bound yeastcells were eluted with 6 mL PBS buffer.

[0207] For yeast selections using the capillary washing device (CWD) theyeast cell mixture and 100 μl streptavidin coated paramagnetic beads(Dynal M280) were blocked in 1 mL 2%MPBS for 1 hour. The paramagneticbeads were resuspended in 1 mL of yeast cells suspension and gentlyrotated for 1 hour at room temperature in an eppendorf tube. Afterincubation of the yeast cells with the streptavidin coated paramagneticbeads the mixture was introduced into the capillary (1 mL was used toload one capillary in five 200 μL steps) of the CWD. After automatedwashing and resuspension of the yeast bead mixture, a final wash withPBS was performed and the yeast/beads complex was collected by adjustingthe magnet.

[0208] Use of the two selectable markers allowed discrimination of thespecific yeast (which are able to grow on minus tryptophan selectiveagar plates) from the none specific yeast cells (which are able to growon minus leucine selective agar plates). The number of colony formingunits (CFUs) for each titre was tallied.

[0209] The enrichment factor was calculated as the ratio of specificyeast cells before and after selection divided by the ratio of the nonspecific yeast before and after selection. Table 1: Model enrichment ofFab displaying yeast cells: Detection by magnetic bead selection. TABLE1 Model enrichment of Fab displaying yeast cells: Detection by magneticbead selection. Ratio^(a) Total cells^(b) Enrichment^(c) Recovery(%)^(d) Kingfisher 1/100  ˜10⁷ 288,000 12.8 1/1000 ˜10⁸ 1,100,000 6 1/10000 ˜10⁹ 400,000 10.7 Capillary Washing Device (CWD) 1/100  ˜10⁷76,000 4.7 1/1000 ˜10⁸ 41,000 6  1/10000 ˜10⁹ 10,000 5.3 MACS 1/1000 10⁷100 12

[0210] As shown in Table 1 specific yeast cells displaying a Fabantibody to streptavidin can be enriched by between 2 and 6 orders ofmagnitude over non relevant yeast cells by one round of selection in anautomated magnetic bead selection device such as Kingfisher, capillarywashing device or magnetic activated cell sorting (MACS).

Example 5 Preferential Enrichment of Fab-Displaying Yeast Cells:Detection by Flow Cytometry

[0211] As an alternative to the magnetic bead detection method ofExample 4 above, enrichment of an antigen-specific Fab antibody over anexcess of non relevant yeast cells was demonstrated usingfluorescence-activated cell sorting (FACS) techniques.

[0212] The Fab-displaying yeast cells, EBY100 pTQ3-F2 (with a tryptophanauxotrophic selectable marker), were mixed with the nonspecific yeastcells, EBY 100 (pUR3867-PH1) carrying a Leu auxotrophic marker, atratios of 1:100, 1:1000 and 1:10,000. The yeast cell mixture wasincubated with 1 μM streptavidin-FITC (Dako, Carpinteria, Calif.) andallowed to equilibrate for 30 minutes at room temperature.

[0213] Three thousand cells were sorted by flow cytometry, and 6.5% ofcells were collected with the highest fluorescent signal. Yeast cellsbefore and after selection were plated on SDCAA+G agar plates andSD-Leu+G agar plates and the number of CFUs determined. The enrichmentfactor was calculated as the ratio of the output ratio divided by theinput ratio of EBY100 pTQ3-F2 and EBY100 pUR3867-PH1.

[0214] After one round of FACS, EBY100 pTQ3-F2 was enriched over EBY100pUR3867-PH1 ten-fold (data not shown). TABLE 2 Enrichment factorsdetermined using FACS. Initial purity^(a) (%) Sorted purity^(b) (%)Enrichment factor^(c) 1.6 85 52 0.79 29 36 0.02 5.2 212

Example 6 Batch Transfer of a Phage Display Antibody Library to aMulti-Chain Eukaryotic Display Vector

[0215] As a demonstration of the utility of the phage display/eukaryoticdisplay transfer system to transfer a phage display peptide library enmasse to a multi-chain eukaryotic display vector of the presentinvention, a phage display Fab library prepared using techniques knownin the art was transferred to the multi-chain yeast display vector pTQ3produced as described in Example 1 above.

[0216] To transfer the phage display repertoire into the multi-chainyeast display vector the single excision/insertion transfer processdescribed earlier and illustrated in FIG. 1 was used (see also Example1).

[0217] A 50 mL culture of TYAG (TY, ampicillin 100 μg/mL, glucose 2%)was inoculated with 10 μL of a glycerol stock from one round ofselection on streptavidin of a naive Fab library cloned into phage (deHaard, H. et al., 1999). The culture was grown overnight at 37° C. andplasmid DNA was prepared (QIAGEN plasmid purification system, Qiagen,Valencia, Calif.).

[0218] The Fab antibody repertoire was digested with ApaLI and NotI andFab antibody fragments of approximately 1.5 kb were recovered andpurified by extraction from a 1.0% TBE ethidium bromide agarose gel(QIAEX gel extraction kit, Qiagen, Valencia, Calif.).

[0219] Similarly, the multi-chain yeast display vector pTQ3 was digestedwith ApaLI and NotI and a fragment of approximately 4.6 kb was purifiedby extraction from a 1.0% TBE ethidium bromide agarose gel.

[0220] Ligation of the Fab antibody inserts recovered from the Fablibrary into the pTQ3 plasmid digested with ApaLI and NotI was performedat a ratio of 4:1 (insert-vector) using 1 μg Fab fragments and 0.7 μgpTQ3 vector in a 100 μL reaction overnight at 16° C. The ligation mixwas purified by phenol, chloroform and isoamyl alcohol (PCI) extractionsand subsequently precipitated with 100% ethanol.

[0221] The purified ligation mix was transformed into E. coli strain TG1(Netherlands Culture Collection of Bacteria, PC 4028, Utrecht, NL) byelectroporation using a BioRad Pulser (BioRad, CA) at 2.5 kV, 25 mF and200 W. The library was plated on 2×TY agar plates (16 g/Lbacto-tryptone, 10 g/L yeast extract, 5 g/L NaCl, 15 g/L bacto-agar)containing ampicillin at 100 μg/mL and 2% w/v glucose (TYAG plates).After overnight growth at 37° C. the repertoire was recovered in 2×TYmedium plus ampicillin at 100 μg/mL by flooding the plates, and frozenin aliquots in 15% (w/v) glycerol.

[0222] The library contained 5.6×10⁶ independent clones. 15 μL of alibrary suspension of 5.4×10¹⁰ cells/mL was used to inoculate 100 mL ofTYAG, and the culture was grown overnight at 37° C. Plasmid DNArecovered as described above.

[0223] The intermediate pTQ3-Fab repertoire was then digested with AscIand with SfiI. A fragment of approximately 6.1 kb was purified asdescribed above. Source vector pTQ3 was similarly digested with AscI andSfiI and a fragment of approximately 1150 bp was purified.

[0224] The purified 1150 bp fragment above was ligated with the pTQ3-Fabrepertoire digested with AscI and SfiI in a ratio of 6:1 (insert-vector)using 1.6 μg insert and 1 μg vector. The ligation mix was purified andtransformed into E. coli strain TG1 as described above to give a finalpTQ3-Fab library of 1×10⁶ independent clones.

[0225] The library was recovered from plates as described above, and 10mL was inoculated in 50 mL TYAG and grown overnight at 37° C. PlasmidDNA was prepared from pTQ3-Fab library and transformed into yeast strainEBY10O by the method of Gietz, D. et al., (1992) to give a final librarysize in yeast of 2×10⁶ independent yeast clones.

Example 7 Selection of Batch Transferred Eukaryotic Display Fab LibraryDetection by Magnetic Bead Selection

[0226] To demonstrate that a yeast display Fab library can undergoselection from a population of yeast cells displaying a diverserepertoire of Fab antibodies, multiple selection experiments wereperformed using an automated magnetic bead selection device.

[0227] The yeast repertoire prepared in Example 6 was grown at 30° C. inSDCAA+G, and antibody expression was induced with galactose (as inExample 4). The pool of yeast cells was incubated for 1 hour with 100 μLstreptavidin paramagnetic beads (Dynal M280, Dynal Biotech, Oslo,Norway) in an end volume of one mL 2% MPBS.

[0228] After incubation of the yeast-bead mixture, the cell-beadcomplexes were washed for 11 cycles in 2% MPBS by transferring thecomplexes from one well to the next well in the automated magnetic beadselection device. After the 2% MPBS washing, two more washing steps wereperformed with PBS. In the last well of the automated magnetic beadselection device, the cell-bead complexes were resuspended in 1 mL PBSand the yeast colony titres before and after selection were determinedby plating on SDCAA+G agar plates. The selected yeast cells were thenused to inoculate a fresh culture of 10 mL SDCAA+G and a second round ofselection was performed as above.

[0229] The percentage of positive and negative clones was determined byyeast whole cell ELISA after the first round of selection and after thesecond round of selection. Cells were grown and induced in a 96 wellplate (Corning Costar, Cambridge, Mass.) in 100 mL SDCAA plus 2% (w/v)galactose.

[0230] After induction, cells were washed one cycle with PBS and dividedequally onto two plates for detection of antigen binding and heavy chaindisplay. In one plate the cells were resuspended in 100 μL 2% MPBScontaining anti-streptavidin-HRP (0.87 μg/mL) for detecting antigenbinding. The cells of the second plate were resuspended in 100 μL 2%MPBS containing anti-c-Myc (1 μg/mL) for detecting heavy chain display.

[0231] After one hour incubation the cells were washed two cycles withPBS and determination of specific binding occurred by resuspending thecells in 100 μL TMB solution. After color development, the reaction wasstopped by adding 50 μL 2N sulfuric acid. Cells were pelleted bycentrifugation, and 100 μL of supernatant was transferred to a flexible96 well plate (Falcon, B D Biosciences, Bedford, Mass.) and theabsorbance at 450 nm recorded. For heavy chain detection, 100 μL 2% MPBScontaining rabbit anti-mouse-HRP (1:1000) was added to each well. Aftera one hour incubation, the cells were washed for two cycles and heavychain display was detected as described above. The results are presentedin Table 3. TABLE 3 Yeast Fab library selection. Round Input OutputRatio % Binders 1 4.9 × 10⁹ 1.1 × 10⁵ 2.7 × 10⁻⁵ 20 2 3.0 × 10⁹ 3.3 ×10⁵ 1.1 × 10⁻⁴ 100

[0232] After one round of selection 20% of the yeast clones screened forantigen binding were found to be positive, after the second round ofselection the number of antigen reactive yeast clones was 100%.

Example 8 Affinity Selection of Anti-Streptavidin Displaying YeastCells: Detection by Flow Cytometry

[0233] In another affinity discrimination experiment, clones EBY100pTQ3-F2 and EBY100 pTQ3-A12/pESC contain an empty vector pESC(Stratagene, La Jolla, Calif.) carrying the Leu auxotrophic marker. Theanti-streptavidin antibody F2 has an affinity of 54 nM as determined byplasmon resonance (BIAcore) and the anti-streptavidin antibody A12 hasan affinity of approximately 500 μM. These two clones were grownovernight and diluted to OD₆₀₀ of 1.0 in SDCAA plus 2% (w/v) galactoseand grown for 48 hours at 20° C. The high affinity antibody containingclone (EBY100 pTQ3-F2) and the low affinity antibody containing clone(EBY100 pTQ3-A12/pESC clones were mixed at a ratio of approximately1:100. Using the different selectable markers present in each cloneallowed discrimination of EBY100 pTQ3-A12/pESC (which are able to growon minus tryptophan, minus leucine selective agar plates) from EBY100pTQ3-F2 (which can only grow on minus tryptophan selective agar plates).The cell mixture was labeled as previously except with a serial dilutionof streptavidin-FITC of 500 nM, 100 nM, 50 nM, 25 nM and 10 nM. Cellswere sorted by flow cytometry in an EPIC ALTRA (Beckman Coulter,Fullerton, Calif.) on the basis of both LC display and antigen binding.The sorting rate was set at 2000 cells/sec and the sorting gate was setto collect 1% of the cell population with the highest ratio of FITC toPE 9typical FACS histogram is shown in FIG. 7). The input and outputcells after selection at different antigen concentrations were titratedon selective plates and the number of colonies were tallied to calculatethe enrichment factor and percentage recovery of the higher affinityclone (Table 4). These results demonstrate that the higher affinityclone can be preferentially recovered by flow cytometric sorting andthat the optimum antigen concentration is between 100 nM and 25 nM for amixture of two antibodies of K_(d=)54 nM and K_(d) of approximately 500nM. TABLE 4 Affinity discrimination of two yeast displayed Fabantibodies of different affinities. Titre Titre (-Trp) Percent- AntigenTitre (-Trp/-Leu) −(-Trp/-Leu) age Enrich- (nM)^(a) (-Trp)^(b)Fab-A12^(c) Fab-F2^(d) Fab-F2^(e) ment^(f) Input 3.2 × 10⁷ 3.7 × 10⁷ 4.7× 10⁵ 1.3 Output 500 nM    9 × 10³ 8.9 × 10³ 100 1.1 0.9 100 nM  1.3 ×10³ 7.9 × 10² 5.6 × 10² 71 56 50 nM 2.4 × 10³ 1.2 × 10³ 1.2 × 10³ 102 8025 nM 1.2 × 10³ 9.1 × 10² 3.2 × 10² 35 27 10 nM 1.53 × 10³  1.49 × 10³  45 3 2.3

Example 9 Construction of Yeast-Displayed Libraries Diversified byError-Prone PCR

[0234] To demonstrate the ability to generate novel multi-chain displayvector libraries the Fab antibody F2, specific for streptavidin, wassubjected to error prone PCR. Separate LC, HC and total Fab antibodywere cloned into the yeast display vector. Error-prone PCR was performedin the presence of 2.25 mM MgCl₂ and 0.375 mM MnCl₂ for 30 cycles.Purified products were cloned into pTQ3 yeast display vectors as aApaL1/Asc1 fragment, SfiI1/NotI fragment or ApaL1/NotI fragmentcorresponding to the LC, a HC and a whole Fab fragment as in Example 2.The ligation mix was transformed into E. Coli and grown on selectiveagar plates containing 100 μg/mL ampicillin to give a LC repertoire of5×10⁶ designated pTQ3F2-LCeP, a HC repertoire of 5.6×10⁸ designatedpTQ3F2-HCeP and a whole Fab repertoire of pTQ3F2-FabeP. The repertoireswere harvested and an inoculated of 200 mL (sufficient to encompass atleast 10 times the library diversity) was made. Plasmid DNA was isolatedfrom a 200 mL culture and transformed into the yeast strain EBY100 asdescribed in Example 2. The resulting repertoires were designatedEBY100-pTQ3F2-LC^(ep) (size=5×10⁶): EBY100-pTQ3F2-HC^(ep)(size=1.7×10⁶); EBY100-pTQ3F2-FabeP (size=10⁶). The mutation frequencyat the nucleotide level was 1.5% for the LC and 0.8% for the HC. Themutation frequency at the amino acid level was 3% for the LC and 1.3%for the HC.

Example 10 Affinity Selection of Anti-Streptavidin Displaying Yeast CellLibrary Detection by Flow Cytometry

[0235] To demonstrate affinity selection of a multi-chain yeast displaylibrary overnight cultures the libraries EBY100-pTQ3F2-LC^(ep):EBY100-pTQ3F2-HCeP and EBY100-pTQ3F2-FabeP were prepared as in Example 2and diluted to OD₆₀₀ of 1.0 in selective media containing SDCAA plus 2%(w/v) galactose and grown for 48 hours at 20° C. The repertoire waslabeled with anti-HA mAb (25 μg/mL) for 1 hour at room temperaturefollowed by a second incubation step with rabbit anti-mouse Ig-FITC(1:40 dilution) and 6 nM streptavidin PE for 1 hour at room temperature.Cells were washed once with 0.5 mL PBS following each incubation stepand after the final wash cells were kept on ice to prevent antigendissociation. Samples were sorted in an EPIC ALTRA flow cytometer with asorting rate of 2000 cells/sec. The first sorting round was done inenrichment mode and the sorting gate was set to collect a population ofcells gated on the basis of both LC display and antigen binding. Thepercentage of cells collected was decreased with successive rounds ofselection to account for the decreasing diversity of the repertoire(FIG. 8A). The collected cells were then grown up to an OD₆₀₀ of 1.0 at30° C. in SDCAA plus (w/v) glucose followed by induction with galactoseas in Example 2. Selection was repeated for rounds 2 and 3 which wereperformed in purity mode with decreasing sorting gates (Table 5).Polyclonal FACS analysis was also performed at different antigenconcentrations, and FACS histograms of both LC display and antigenbinding activity are shown in FIG. 8B. TABLE 5 Selection of error-pronerepertoires. Total Ag % Cells % Ag Round Repertoire Size Sampled (nM)Strategy FACS mode collected Binding R1 pTQ3F2LC^(ep)     5 × 10⁶ 6 ×10⁶ 6 FACS Enrichment 6.0 40 R2 ″ ″ 4 × 10⁶ 6 FACS Purity 1.4 70 R3 ″ ″4 × 10⁶ 6 FACS Purity 0.2 75 R1 pTQ3F2HC^(ep)       1.7 × 10⁶ 3 × 10⁸ —Kingfisher — — 23 R2 ″ ″ 2 × 10⁶ 6 FACS Purity 1.4 72 R3 ″ ″ 4 × 10⁶ 6FACS Purity 0.5 62 R1  pTQ3F2Fab^(ep)   10⁶ 5 × 10⁶ 6 FACS Enrichment5.0 18 R2 ″ ″ 4 × 10⁶ 6 FACS Purity 1.4 60 R3 ″ ″ 5 × 10⁶ 6 FACS Purity0.2 73

Example 11 Analysis of Selected Fab Antibodies

[0236] The yeast clones retrieved from the affinity selection of therepertoires EBY100-pTQ3F2-LC^(ep); EBY100-pTQ3F2-HC^(ep);EBY100-pTQ3F2-Fab^(ep) were affinity screened to quantitate theimprovement in affinity over the starting wild-type antibody. Theselected antibodies were also sequenced to determine the mutations thatcorrelate with improved affinity.

[0237] Yeast colonies were picked and resuspended in 25 μL of lyticasesolution (2.5 mg/mL; Sigma, St. Louis, Mo.) for 1 hour at 37° C. afterwhich 2 μL was taken and used in a PCR reaction. Separate LC and HC wereamplified and sequenced using and ABI-PRISM sequencer. Mutations fromwild-type were determined using sequence alignment and are shown inTable 6. TABLE 6 Overview of mutated Fab antibodies selected from errorprone repertoires by FACS. Sequence Sequence Normalized Repertoire RoundClone V_(L) V_(H) FACS signal¹ wt-F2 1.00 F2 LC^(ep) R1 R1C9 F62I / ″ R1R1H8 S2P, D85V / 1.42 ″ R1 R1H10 H34R, Y96H / 1.15 ″ R2 R2H8 S2P, D85V /1.23 ″ R2 R2H10 H34R, Y96H / 1.23 ″ R2 R2A7 no a.a mut. / 0.95 ″ R3 R2H8S2P, D85V / 1.9 F2 HC^(ep) R3 R3H4 / H53R ″ R3D2 / H53R; S62A F2Fab^(ep) R2 R2D3 H34R no a.a mut. 1.65 ″ R2 R2G4 V11A, H34N, V58A, P40L0 S67P, L95I ″ R3 R3B1 Y96F P40L 1.78 ″ R3 R3H1 ″ A23V, S65R 1.50 ″ R3R3E1 S2P, D85V K14E 1.56 ″ R3 R3G4 ″ H53R, A84T 1.70 ″ R3 R3F1 Q1L,K45R, L95V no a.a mut. 1.60 ″ R3 R3A3 H34R no a.a mut. 1.65 ″ R3 R3H3H34R, Q79R Q3R 1.62

[0238] The off rate of the selected Fabs was determined by measuring thedissociation rate in FACS as the decrease in fluorescence signal overtime; the clone, R2H10 gave the greatest improvement in affinity (10.7fold, 3.2 nM). This dissociation rate was fit to a exponential decaymodel and the k_(d) calculated. Yeast cells were labeled with bothanti-HA to detect the LC and also for antigen with streptavidin PE.Yeast cultures were grown an induced as described Example 2 andapproximately 2×10⁷ cells were collected and washed with PBS. The cellswere then incubated with 100 μL anti-HA Mab (20 μg/mL) for 1 hour andthen washed with 0.5 mL of PBS. The cells were then incubated withrabbit anti-mouse FITC (1:40) and streptavidin PE (1:40 dilution of 1μg/mL stock) for 1 hour on ice. The cell pellet was then resuspended inan excess of non-fluorescent ligand at room temperature. Theconcentration of non-fluorescent label was taken so that it was 10-100fold in excess of the molar concentration of yeast displayed Fabantibody assuming there are approximately 100,000 copies of a Fabantibody per yeast cell. The decrease in fluorescence intensity wasmonitored for 1.5 mins. to 30 mins. by flow cytometry. Unlabeled yeastcells were used to set the background fluorescence. The k_(d) was thencalculated by fitting the dissociation rate to a model of exponentialdecay from which the k_(d) was calculated. FIG. 10c shows the off ratedetermination by FACS for clones wild-type F2, and mutants R2E10, R3B1and R3H3.

[0239] The affinity of soluble Fabs was determined by subcloning theselected Fab antibodies into the E. coli expression vector pCES 1 as inExample 2. Soluble Fabs were purified and affinity tested via BIAcore(de Haard, H. et al.). The affinity of selected Fabs is shown in Table7. TABLE 7 Characterization of affinity improved Fab fragments.Mutations FACS^(c) Biacore^(c) Mutations Variable k_(d) k_(d) k_(a)K_(D) fac- Clone Library Variable LC^(a,b) HC^(a,b) (10⁻⁴ s⁻¹) (10⁻³s⁻¹) (10⁴ M⁻¹ s) nM tor wt-F2 / none none 2.2 ± 1.0 1.52 ± 0.15 4.51 ±0.01 34 / R2H10   LC e.p. H34R, Y96H none 0.5 ± 0.1 0.18 ± 0.01 5.69 ±0.02 3.2 10.7 R3A9 ″ S2P, D85V none 1.3 ± 0.1 1.53 ± 0.57 7.84 ± 0.0819.5 1.7 R3H4   HC e.p. none H53R 1.9 ± 0.7 N.D. N.D N.D N.D . . R3D2none H53R, S62A 1.6 ± 0.6 N.D. N.D. N.D N.D . . R2D3   fab e.p. H34R 1silent mut. 2.1 ± 0.3 1.04 ± 0.10 5.76 ± 0.08 18.1 1.9 R3H1 ″ Y96F A23V,S65R 1.0 ± 0.4 0.28 ± 0.04 3.25 ± 1.14 8.7 3.9 R3G4 ″ S2P, D85V H53R,A84T 3.5 ± 1.1 2.37 ± 0.25 10.9 ± 1.13 21.7 1.6 R3B1 ″ Y96F P40L 0.9 ±0.2 0.22 ± 0.05 4.00 ± 1.30 5.5 6.3 R3E1 ″ S2P, D85V K14E 2.1 ± 1.2 1.03± 0.10 7.64 ± 0.95 13.5 2.5 R3H3 ″ Q79R Q3R 2.0 ± 1.0 1.04 ± 0.04 11.3 ±2.64 9.2 3.7

Example 12 Rapid Selection of Yeast Displayed Fab Repertoire Using aCombination of Kingfisher and FACS Selection

[0240] In order to speed up the affinity selection of yeast displayedrepertoires and also to develop methodologies which allow for theselection of larger repertoires in excess of 10⁸, a combination of bothKingfisher as the first round of selection (as in Example 4) and FACSfor the latter rounds of selection (as in Example 5) was used. The LCrepertoire constructed in Example 9 was grown overnight and antibodyexpression was induced as in Example 2. The yeast cell population wasincubated with streptavidin coated magnetic particles and selected withKingfisher as in Example 4. In parallel, the same repertoire wasselected by FACS as in Example 5. The pool of yeast cells from the round1 selection campaigns using Kingfisher and FACS was grown overnight andantibody expression induced as in Example 5. Yeast cells were labeled asin Example 2 and selected by FACS as the second round. Analysis of theselected pools of yeast displaying Fabs was performed using polyclonalFACS (see Example 10). The percentage of antigen binding cells can beseen to increase faster when Kingfisher is used as the first round ofselection in preference to FACS (FIG. 8d).

Example 13 Construction of an Ig Heavy Chain Eukaryotic Display VectorpTQ5-HC

[0241] As a demonstration of an alternate embodiment of the multi-chaineukaryotic display vector of the present invention (specifically amulti-chain eukaryotic display vector wherein the chains of themulti-chain are encoded on separate vectors, thus forming separatecomponents of a vector set), a yeast display vector effective in a hostyeast cell transformed with the vector of expressing, transporting, anddisplaying an Ig heavy chain fragment was constructed as one vector of amatched vector set.

[0242] An HC fragment display vector was constructed by further alteringthe vector pTQ3 produced according to Example 1. Display vector TQ3 wasdigested with BseRI, thus identifying a designed restriction site of thevector positioned in each of the two tandem GAL1 promoters (see Example1, SEQ ID NO: 5 designated bases 990-995). A 942 bp fragment, whichspans one of the cloning sites of the multi-chain display vector (FIG.3), was removed and the remaining 4,868 bp vector backbone was gelpurified using techniques known in the art (specifically via GFX PCR andGel Band Purification Kit, Amersham-Pharmacia, Piscataway, N.J.). Thevector backbone was re-ligated and transformed into E. coli. Theresultant vector, designated “pTQ5”, was verified using by restrictionanalysis.

[0243] The HC for the anti-streptavidin Fab antibody F2 was restrictiondigested from pTQ3-F2 as a 709 bp SfiI/NotI fragment, purified, andcloned into SfiI/NotI digested vector pTQ5. The resultant HC displayvector was designated “pTQ5-HC” (FIG. 9).

[0244] Later modifications were made to this vector by inserting a 6×Histag for purification of soluble Fab antibodies and by repositioning thestop codon (TAA) at the end of the myc tag, before the PacI site, toeliminate superfluous amino acids. Other modifications have included theremoval of an endogenous XbaI restriction site within the Trp selectivemarker by site directed mutagenesis. This was done to facilitate cloningand manipulation of lead antibodies from the CJ library set (DyaxCorporation, Cambridge, Mass.).

Example 14 Eukaryotic Host Cell Expression of an Ig Heavy ChainEukaryotic Display Vector: HC Expression in a Haploid Yeast Cell

[0245] To demonstrate the utility of independent vectors of amulti-chain eukaryotic display vector set, a yeast display vector (of avector set) encoding an Ig heavy chain fragment was inserted into aeukaryotic host cell, and the transformed host cell grown underconditions suitable for expression of the heavy chain component of an IgFab.

[0246] The yeast strain EBY 100 (InVitrogen, Carlsbad, Calif.) wastransformed with vector pTQ5-HC (of Example 13), and separately withpTQ5 as a control, following transformation procedures previouslydescribed. The successful transformants, designated EBY100 pTQ5-HC andEBY100 pTQ5 respectively, were cultured overnight at 30° C. in 10 mLSDCAA+G.

[0247] The next day cultures were centrifuged and the pelleted yeastcells were resuspended in 10 mL SDCAA plus 2% (w/v) galactose to anOD₆₀₀ of 1. Cell cultures were then grown for 24 hours at 20° C. toinduce expression of the Aga2p heavy chain fusion product. Cells werecentrifuged and washed twice in 1 mL sterile water and transferred to aneppendorf tube.

[0248] Cell pellets were resuspended in 200 mL of SDS-PAGE sample bufferplus DTT, and glass beads (425-600 micron) were applied to just belowthe meniscus. The cell and bead suspension was vortexed 4 times for 1minute keeping the suspension on ice between vortexing. The supernatantwas transferred to a fresh tube and heated to 100° C. for 5 minutes.

[0249] Protein samples were separated on a SDS-PAGE gel and transferredto a nitrocellulose membrane for western blotting. Detection of theAga2p-HC fusion polypeptide was performed using an anti-c-Myc monoclonalantibody conjugated to HRP (1 μg/mL, Roche Molecular Biochemicals,Indianapolis, Ind.). Immunodetection was by enhanced chemilluminescence(Amersham-Pharmacia, Piscataway, N.J.). The 45 kD Aga2p-HC fusionpolypeptide approximately was detected. No detectable Aga2p-HC fusionproduct was detected in the (empty) control vector clone EBY100 pTQ5(FIG. 10).

Example 15 Eukaryotic Host Cell Display of an Ig Heavy Chain EukaryoticDisplay Vector: HC Display on the Surface of a Haploid Yeast Cell

[0250] To demonstrate the ability of a vector from a multi-chaineukaryotic display vector set to display the anchored chain of amulti-chain polypeptide on the surface of a haploid eukaryotic cell, ayeast display vector (of a vector set) encoding an Ig heavy chainfragment was inserted into a eukaryotic host cell, and the transformedhost cell was grown under conditions suitable for expression and displayof the heavy chain component of an Ig Fab.

[0251] EBY100 pTQ5-HC (from Example 14) was grown, and antibodyexpression was induced as above. HC expression was induced by 48 hoursof growth with shaking at 20° C. Yeast samples were centrifuged and thecell pellet resuspended in PBS containing 1 mg/mL BSA. Two of thesamples were again centrifuged and the cell pellets separatelyresuspended in either 100 μL of anti-human C_(H)1 (25 μg/mL; Zymed, SanFrancisco, Calif.) followed by incubation for 1 hour at roomtemperature. The cells were pelleted and washed once with 0.5 mL ofPBS/1% (w/v) BSA. Cell samples were then incubated with rabbitanti-mouse FITC (1:50 dilution; Dako, Carpinteria, Calif.) for 1 hour inthe dark.

[0252] To detect antigen binding, cells were labeled withstreptavidin-FITC (1:25 dilution; Dako, Carpinteria, Calif.) in PBS/1%(w/v) BSA and incubated in the dark at room temperature for 1 hour. Allsamples were centrifuged and the cell pellets washed once with 0.5 mLPBS and then resuspended in 500 mL of PBS.

[0253] The presence of cell surface bound HC-antigen binding wasdetected by flow cytometry. Cells prior to induction showed no displayof heavy chain or functional streptavidin binding. After induction of HCexpression, yeast cells could be detected displaying heavy chain onlybut no functional streptavidin binding could be detected as expected(FIG. 11).

Example 16 Construction of an Ig Light Chain Eukaryotic Display VectorpTQ6-LC

[0254] A light chain yeast display vector was constructed to provide amulti-chain eukaryotic display vector set, i.e., when used inconjunction with the heavy chain yeast display vector described above(see Example 13, supra).

[0255] A LC yeast expression vector was constructed by amplifying afragment containing the anti-streptavidin LC fused to the HA epitope tagand Aga2p signal sequence. The amplification product was gel purifiedusing a GFX PCR and Gel Band Purification Kit (Amersham-Pharmacia,Piscataway, N.J.), and digested with HindIII and Pmel. The 783 bp LCfragment was purified on a 1.2% TAE-agarose gel together with the 4,323bp vector backbone of a HindIII/Pmel-digested pYC6/CT vector(InVitrogen, Carlsbad, Calif.). The LC fragment and pYC6/CT vector wereligated together and the ligation mix was transformed into E. colistrain TG1. The resultant LC expression vector was designated “pTQ6-LC”(FIG. 12).

Example 17 Eukaryotic Host Cell Expression of an Ig Light ChainEukaryotic Display Vector: Soluble LC Expression in a Haploid Yeast Cell

[0256] To demonstrate the utility of independent vectors of amulti-chain eukaryotic display vector set, a yeast display vector (of avector set) encoding an Ig light chain fragment was inserted into aeukaryotic host cell, and the transformed host cell grown underconditions suitable for expression of a soluble light chain component ofan Ig Fab.

[0257] Yeast strain W303-1B (a/alpha ura3-1/ura3-1 leu2-3,112/leu2-3,112trpl-1/trpl-1 his3-11,15/his3-11,15 ade2-1/ade2-1 can1-100/can1-100),obtained from P. Slonimski, was transformed with pTQ6-LC (of Example 16)and separately pYC6/CT as a control, following transformation procedurespreviously described. The successful transformants, designated W303pTQ6-LC and W303 pYC6/CT respectively, were cultured overnight at 30° C.in 10 mL SD-G plus 300 μg/mL Blasticidin® (SD-G+Bls).

[0258] The next day cultures were centrifuged and the pelleted yeastcells resuspended in 10 mL SD+Bls plus 2% (w/v) galactose to an OD₆₀₀ of0.4. Cell cultures were then grown for 24 hours at 20° C. to induceexpression of the soluble light chain polypeptide. Cells werecentrifuged and the supernatants were concentrated ten fold using acentrifugal filter unit (CENTRICON YM-10; Millipore, Bedford, Mass.).

[0259] Cell pellets were washed and resuspended in breaking buffer (50mM sodium phosphate, pH 7.4, 1 mM EDTA, 5% (w/v) glycerol plus proteaseinhibitor cocktail; Roche Molecular Biochemicals, Indianapolis, Ind.) toan OD₆₀₀ of 50, and glass beads (425-600 micron) were applied to justbelow the meniscus. The cell and bead suspension was vortexed 4 timesfor 1 minute keeping the suspension on ice between vortexing. Thesupernatant was transferred to a fresh tube and an aliquot was heated to10° C. for 5 minutes in SDS-PAGE sample buffer plus DTT.

[0260] Protein samples were separated on a SDS-PAGE gel and transferredto a nitrocellulose membrane for western blotting. Detection of the LCpolypeptide was performed using an anti HA monoclonal antibody (1 μg/mL)in combination with a rabbit anti-mouse conjugated to HRP ({fraction(1/1000)}). Immunodetection was by enhanced chemilluminescence(Amersham-Pharmacia, Piscataway, N.J.). Polypeptide products of 30 kDand 60 kD were detected in the culture supernatant. No detectable LCproduct could be detected in the empty vector control W303 pYC6/CT (FIG.13).

Example 18 Surface Display of a Multi-Chain Polypeptide on a EukaryoticHost Cell: The Product of Cellular Fusion of a Haploid Host Cell Pair

[0261] To demonstrate the operability of the novel process fordisplaying a biologically active multi-chain polypeptide on the surfaceof a diploid eukaryotic cell via the cellular fusion of two haploideukaryotic cells, each possessing a different vector from a matchedmulti-chain eukaryotic display vector set, haploid yeast cellscontaining a vector expressing a soluble Ig light chain fragment weremated to haploid yeast cells containing a vector expressing anddisplaying an Ig heavy chain-anchor fusion polypeptide to produce adiploid yeast cell that displays a functional Fab polypeptide on thesurface of the host cell.

[0262] Yeast clones W303 pTQ6-LC (from Example 17) and EBY100 pTQ5-HC(from Example 14) were grown on agar plates supplemented with eitherBlastocidin® (InVitrogen, Carlsbad, Calif.; 300 μg/mL; SD+G+Bls agarplates) or tryptophan drop out medium (SD-Trp+G agar plates). Theseplates were then replica plated onto double selective plates containingsynthetic defined medium for tryptophan dropout plus 300 μg/mLBlasticidin® (SD-Trp+G+Bls). The resulting cell layer of diploid yeastcells was streaked to single colonies. Seven Trp+/Bls^(R) colonies wereselected and grown overnight with shaking at 30° C. in 100 mLSD+G-Trp+Bls in 96-wells plate.

[0263] The next day, the culture was centrifuged and the pelleted yeastcells were resuspended in either 10 mL SD-Trp+Bls plus 2% (w/v)galactose or 10 mL YP medium+Bls plus 2% (w/v) galactose for 24 hours at20° C. Cells were washed in PBS and divided equally onto three 96 wellplates. Cells of the first plate were resuspended in 100 μLstreptavidin-HRP (0.87 μg/mL), cells of the second plate wereresuspended in 100 μL anti-c-Myc-HRP (1 μg/mL), and cells of the thirdplate were resuspended in 100 pL anti-HA (1 μg/mL) and additionallylabeled with a rabbit anti-mouse-HRP ({fraction (1/1000)}).

[0264] Yeast whole cell ELISA was performed (as in Example 7) and FACS(as in Example 15) was performed to detect antigen binding and HCdisplay. All diploids tested bound to streptavidin and displayed lightchains in whole cell ELISA (FIG. 14) and FACS (FIGS. 15A-C).Specifically, streptavidin binding activity was detected on diploidyeast cells displaying combinatorially assembled Fab antibody (DiploidLC/HC) on their surface whereas haploid parents expressing either LConly (W303 pTQ6-LC) or HC only (EBY100 pTQ5-HC) showed no bindingactivity. Standard haploid yeast cells displaying a Fab antibody (EBY100pTQ3-F2) showed streptavidin binding activity. Also (as expected) thehaploid parent yeast cell expressing only LC (W303 pTQ6-LC) showed no HCdisplay, while standard haploid yeast cells displaying a Fab antibody(EBY100 pTQ3-F2) showed HC display.

[0265] Five yeast clones were selected for overnight culture at 30° C.in 10 mL SD+G-Trp+Bls with shaking. The next day, cell cultures werecentrifuged and the pelleted yeast cells resuspended in 10 mL SD-Trp+Blsplus 2% (w/v) galactose to an OD₆₀₀ of 0.4 for 24 hours to induce vectorexpression. An alternative protocol involves resuspension in 10 mL of YPmedia plus Blastocidin® plus 2% (w/v) galactose.

[0266] After the 24 hour induction incubation, one aliquot from each ofthe five diploid yeast cultures was pelleted, washed, and resuspended inbreaking buffer to an OD₆₀₀ of 50. Glass beads (425-600 micron) wereadded to just below the meniscus, and the cell-bead suspension vortexed4 times for 1 minute keeping the suspension on ice between vortexing.The supernatant was transferred to a fresh tube and an aliquot washeated to 100° C. for 5 minutes in SDS-PAGE sample buffer plus DTT.

[0267] Protein samples were separated on SDS-PAGE gels and transferredto a nitrocellulose membrane for western blotting. Detection of thelight chain polypeptide was performed using an anti-HA antibody (1μg/mL) in combination with a rabbit anti-mouse conjugated to HRP on onemembrane. Detection of the heavy chain-Aga2p fusion polypeptide wasperformed using an anti-c-Myc antibody directly conjugated to HRP (1μg/mL, Roche Molecular Biochemicals, Indianapolis, Ind.).Immunodetection was by enhanced chemilluminescence (Amersham-Pharmacia,Piscataway, N.J.). The LC product of approximately 30 kD and theHC-Aga2p fusion product of approximately 45 kD were both detected in thediploid yeast lysate (FIGS. 16 and 17). No detectable LC or HC-Aga2pfusion product was detected in control diploid clones harboring the twoempty vectors pTQ5 and pYC6/CT.

[0268] Also after the 24 hour induction incubation, a second aliquotfrom each of the five diploid yeast cultures was analyzed by flowcytometry. 5×10⁶ cells per detection agent were washed one cycle withPBS and the cells were resuspended in 100 μL PBS containing anti-c-Myc(25 μg/mL) for heavy chain detection, 100 μL PBS containinganti-streptavidin-FITC (1:40) for detection of antigen binding and 100μL PBS containing anti-HA (25 μg/mL) for light chain detection. Cellswere incubated for one hour in the dark and than washed again one cyclewith PBS. After washing the cells were resuspended in 100 μL PBScontaining rabbit anti mouse-FITC (1:40) and again incubated for onehour in the dark.

[0269] Cells with anti-streptavidin-FITC were processed during thesecond incubation step because of the one step labeling. Afterincubation, cells were washed for one more cycle and resuspended in 500μl PBS and analyzed by flow cytometry. All five samples were shown tobind to the antigen and to display the HC as well as the LC (FIGS.15A-C).

[0270] After the 24 hour (induction) incubation, a third aliquot fromone of the five diploid yeast cultures was also labeled forimmunofluorescence. 108 cells were resuspended in 100 μL of eitherstreptavidin-FITC (30 μg/mL, Dako) or of a mixture of rabbit anti-humanlambda chain (1:40; Dako, Carpinteria, Calif.) and monoclonalanti-C_(H)1 (25 μg/mL, Zymed, San Francisco, USA). A first sample wasfurther incubated with rabbit anti-FITC (1:40; Dako, Carpinteria,Calif.), and finally with swine anti-rabbit conjugated to FITC (1:20;Dako, Carpinteria, Calif.). A second sample was submitted to a doublelabeling with swine anti-rabbit conjugated to FITC (1:20; Dako,Carpinteria, Calif.) for the light chain and rabbit anti-mouseconjugated to Tetramethylrhodamine isothiocyanate (TRITC, 1:30, Sigma,St. Louis, Mo.) for the heavy chain (FIGS. 18A-C).

[0271] The diploid displayed the light and the heavy chain at the cellsurface and was shown to bind streptavidin, as expected. The haploidparent expressing HC only was stained only by the TRITC labeling of theheavy chain. The haploid parent LC was negative in all the cases.

EXAMPLE 19 Mating Efficiency of a Haploid Host Yeast Cell Pair

[0272] To demonstrate the efficiency of cell fusion of two haploid yeastcells, each possessing a different vector from a matched multi-chaineukaryotic display vector set as a viable process to generate a diploidyeast cell displaying a biologically active multi-chain polypeptide onits surface, mating efficiency was determined for a host yeast cell pairaccording to the present invention. Quantitative determination of theefficiency of the mating reaction was assessed as follows.

[0273] Each haploid parent EBY100 pTQ5 (from Example 14) and W303pYC6/CT (from Example 17) was grown overnight at 30° C. in theappropriate selective medium SD+G-Trp and SD+G+Bls respectively. 3×10⁷cells from the two fresh haploid cultures were mixed and collected on a45 mm nitrocellulose filter (microfill device of Millipore, Bedford,Mass.). The filter was incubated for 4 hours at 30° C. on a nonselective rich medium plate (YPD). Cells were then resuspended in YPDmedium and titrated on the two parental selective media and on thedouble selective medium (which only allows the growth of the diploids)SD+G-Trp+Bls. Spontaneous reversion or resistance was assessed byprocessing each haploid parent separately in the same way and platingthem on the double selective medium without dilution.

[0274] The mating efficiency of the haploid parent EBY100 pTQ5 wascalculated as follows: (the number of diploids growing on SD+G−Trp+Blsminus the number of spontaneous resistant EBY100 pTQ5 growing onSD+G−Trp+Bls) divided by (the total number of cells from the matingreaction showing growth on SD+G−Trp).

[0275] The mating efficiency of haploid parent W303 (pYC6) theefficiency was calculated as follows: (the number of diploids onSD+G−Trp+Bls minus the number haploid cells W303pYC6/CT growing onSD+G−Trp+Bls) divided by (the number of cells on SD+G+Bls).

[0276] 3×10⁷ haploid cells of each mating type produced 1.5×10⁷ diploidcells containing both pTQ5 and pYC6 yeast expression vectors. Matingefficiency results revealed that 51% of haploid parents containing thepTQ5 plasmid formed diploids, and that 64% of haploid parents containingthe pYC6 plasmid formed diploids.

Example 20 Preferential Enrichment of Diploid Yeast Cells Displaying aCombinatorially Assembled Fab Antibody: Detection by Flow Cytometry

[0277] To confirm the ability to select yeast cells displaying anantigen specific Fab antibody over an excess of non-relevant yeastcells, fluorescent activated cell sorting (FACS) was used. A positivediploid yeast cell displaying a combinatorially assembled Fab antibodyspecific for streptavidin was used. The diploid yeast cell carried thephenotypic markers of Trp⁺/Leu⁻/Bls^(R), and was able to grow on minustryptophan and Blastocidin® containing selective agar plates. Thisdiploid was designated the BlsR diploid. A non-relevant yeast diploidcell carrying the phenotypic markers −Trp+/−Leu+was used, and was ableto grow on minus leucine and tryptophan containing selective agarplates. This diploid was designated the Leu+diploid. Both positive(Bls^(R)) and non-relevant (Leu+) diploid yeast cells were grownovernight in media of SD plus 2%(w/v) glucose under selective conditionsof −Trp/+Leu/+Bls media and −Trp/−Leu media respectively. Yeast cultureswere induced in YP media containing galactose at 2% (w/v). Afterdetermining the OD₆₀₀ of the yeast culture and using the conversionfactor of OD₆₀₀ of 1 is equivalent to 4×10⁶ cells/mL, a mix of positiveto non-relevant yeast cells was prepared in an approximate ratio of1:10000. For selection by FACS the yeast cell mixture was labeled with500 nM streptavidin PE and selected as in Example 8. For Kingfisher, theyeast cell mixture was incubated with streptavidin coated beads andselected as in Example 4. For selection by MACS, induced diploids wereincubated for one hour at room temperature with 500 μL streptavidinmicrobeads (Miltenyi Biotec, Cologne, Germany) in 6 mL PBS+2 mM EDTA.The cell/bead mixture was loaded onto a pre-washed LS column (MiltenyiBiotec, Cologne, Germany) in the presence of a magnet and the column wasagain washed twice with PBS+2 mM EDTA. After the removal of the magnet,the cells retained on the column were eluted in 6 mL PBS buffer.

[0278] Yeast cells were recovered and titrated on selective agar platesfor either the Bla^(R) phenotype or the Leu⁺ phenotype. The ratio ofBls^(R)/Leu⁺ colonies before and after selection was used to calculatethe enrichment factor and the percentage recovery of positive yeastcells TABLE 8 Single pass enrichment experiments using MACS, Kingfisherand FACS. Input Output Bla^(R)/ Bla^(R)/ Leu⁺ Bla^(R) Leu⁺ Leu⁺ Bla^(R)Leu⁺ Re- Device diploid diploid (%) diploid diploid (%) covery Enrich.MACS 5 × 10⁸ 4 × 10⁴ 0.008 3 × 10⁵  10⁴ 3.6 25% 465 Kingfisher 6 × 10⁸ 4× 10⁴ 0.0065 600 750  125 1.8%  19230 FACS 2 × 10⁷ 10⁴ 0.05 204  6 3.4ND 68

[0279] In the given example of an antibody specific for streptavidin,Kingfisher was seen to give a higher enrichment factor than MACS.However, the percentage recovery of positive yeast cells wassignificantly lower. Using FACS, an enrichment factor of one order ofmagnitude was observed from one round of selection for ananti-streptavidin Fab antibody.

Example 21 LC and HC Recombination by Cellular Fusion of a Haploid HostCell Pair and Affinity Selection: Detection by Flow Cytometry

[0280] To exemplify the utility of the fusion of two haploid eukaryoticcells, each possessing a different vector from a matched multi-chaineukaryotic display vector set, a haploid yeast cell populationcontaining a vector expressing a plurality soluble Ig light chainfragment variants (i.e., a library of LC variants) is mated to a haploidyeast cell population of opposite mating type containing a vectorexpressing and displaying a plurality of Ig heavy chain-anchor fusionpolypeptide variants (i.e., a library of HC variants) to produce a noveldiploid yeast cell population that displays a plurality of functionalFab polypeptides on the surface of the host cells (i.e., a novel Fablibrary).

[0281] Fab phage display isolates, pre-selected for a target moleculefrom a Fab repertoire, are used to provide the source heavy chain andlight chain components for a batch transfer of the phage display isolategenetic information to a multi-chain yeast display vector set (asdemonstrated in Example 6), using the multi-chain yeast display vectorset (as described in Examples 14 and 17) to provide novel recombinationof light and heavy chain isolates via host cell fusion of two haploideukaryotic cells, each possessing a different vector from a matchedmulti-chain eukaryotic display vector set (as demonstrated in Example18).

[0282] A phage display antibody library (de Haard, H. et al., 1999) wassubjected to one round of selection on streptavidin coated magneticparticles using protocols familiar to those skilled in the art. Thisrepertoire was used as a starting repertoire for transfer into the yeastdisplay system. The input of the library was 5×10¹² phage particles andthe output after one round of selection was 3.75×10⁵ phage particles.

[0283] The HC fragments were isolated from the round 1 selected phagedisplay library as SfiI/NotI fragments and cloned into the Ig heavychain yeast display vector pTQ5, which was digested with SfiI and NotI(Example 13). The ligation mix was transformed into E. coli to give alibrary of 10⁸. This library was then transformed into the yeast strainEBY100 to give a library of 4×10⁷ and designated EBY100-pTQ5-HC_(rep).

[0284] The LC fragments were isolated from the round 1 selected phagedisplay library as ApaL1/AscI fragments and cloned into the Ig lightchain yeast display vector, pTQ6 digested with ApaL1 and AscI (Example16). The ligation mix was transformed into E. coli to give a library of1×10⁸. This library was then transformed into the yeast strain BJ5457 togive a library of 8×10⁷ and was designated BJ5457-pTQ6-LC_(rep). Boththe HC and LC repertoires in yeast contained sufficient diversity tocover the starting repertoire of 3.75×10⁵ in phage. DNA fingerprintanalysis of individual clones showed diverse restriction patternsindicating that different germline segments were represented in theseparate LC and HC libraries.

[0285] In the first mating regime 7.25×10⁸ cells of the LC repertoire(BJ5457-pTQ6-LC_(rep)) were mated 3.4×10⁸ cells of EBY100-pTQ5-F2HCcontaining the single HC specific for streptavidin and derived fromclone F2. The mating conditions were under selective pressure tomaintain both the LC and HC expression plasmids (tryptophan auxotrophyand blastocidin resistance). A library of 1.9×10⁸ diploids was obtainedwith a mating efficiency of 55%. Analysis of individual clones from thislibrary by yeast whole cell ELISA showed 100% of clones displayed a HCand 100% of clones displayed a LC.

[0286] In a second mating regime 3.6×10⁸ cells of the HC repertoire(EBY100-pTQ5-HC_(rep)) were mated with 3×10⁸ cells of BJ5457-pTQ6-F2LCcontaining a single LC specific for streptavidin and derived from cloneF2. The mating conditions were under selective pressure to maintain boththe LC and HC expression plasmids (tryptophan auxotrophy and blastocidinresistance). A library of 8×10⁷ diploids was obtained with a matingefficiency of 27%. Analysis of individual clones from this library byyeast whole cell ELISA showed 89% of clones displayed a HC and all ofthese clones displayed a LC.

[0287] In a third mating regime 2.0×10¹⁰ cells of the HC repertoire(EBY100-pTQ5-HC^(rep)) were mated with 5.6×10⁹ cells of the LCrepertoire (BJ5457-pTQ6-LC^(rep)). The mating conditions were underselective pressure to maintain both the LC and HC expression plasmids(tryptophan auxotrophy and Blastocidin® resistance). A diploid libraryof 4×10⁹was obtained with a mating efficiency of 68%. Analysis ofindividual clones from this library by yeast whole cell ELISA showed 94%of clones displayed a HC and 53% of clones displayed a LC.

[0288] This series of mating experiments shows that large libraries canbe made using the mating of separate repertoires of LC and HC. Theserepertoires comprise diverse V gene germline segments and can beexpressed and displayed on the yeast cell surface. These repertoireswere selected with the antigen streptavidin using Kingfisher (seeExample 7). After two rounds of selection, 97% of clones retrievedshowed antigen binding activity in a yeast whole cell ELISA (see Example7).

Example 22 Construction of LC and HC Repertoires Diversified by ErrorProne PCR

[0289] To demonstrate the fusion of two repertoires of haploid yeastcells, each possessing a different vector from a matched multi-chainvector set, can be used for affinity maturation, a HC repertoirediversified by error prone PCR (Example 9) in pTQ5 (Example 13) and aseparate LC repertoire diversified by error prone PCR (Example 9) inpTQ6 (Example 16) were constructed in yeast haploid cells of oppositemating type.

[0290] The HC repertoire was constructed by amplifying theanti-streptavidin F2 antibody under error prone conditions (Example 9).The amplified fragment was digested with SfiI and NotI, purified andcloned into the HC-only expression vector pTQ5 (Example 13), which hadalso been digested with SfiI and NotI. The resulting ligation mix wastransformed into E. coli to give a library of 7×10⁷. This library wastransformed into the yeast strain EBY100 to give a library of 9×10⁶ andwas designated EBY100 pTQ5-HC*.

[0291] The LC repertoire was constructed by amplifying the LC of theanti-streptavidin F2 antibody under error prone conditions (Example 9).The amplified fragment was digested with ApaL1 and AscI and cloned intothe LC only expression vector (Example 16), which had also been digestedwith ApaL1 and AscI. The resulting ligation mix was transformed into E.coli to give a library of 4×10⁷, and this was subsequently transferredinto the yeast strain BJ5457 to give a library of 1.8×10⁷ (designatedBJ5457 pTQ6-LC*).

[0292] The resulting mutation frequency at the nucleotide level was 0.8%for the HC repertoire and 1.5% for the LC repertoire. These frequenciescorrespond to 1.3% and 3% mutation frequency at the amino acid level,respectively. The haploid cell repertoires EBY100 pTQ5-HC* and theBJ5457 pTQ6-LC* were inoculated with 10 μL and 30 μL of glycerol stockrespectively so that at least 10 copies of each independent clone wasrepresented and grown up overnight in selective media (Example 18).Approximately 1.6×10¹⁰ haploid cells corresponding to BJ5457 pTQ6-LC*and 3×10¹⁰ haploid cells corresponding to EBY100 pTQ5-HC* were mated(Example 19) to give a diploid repertoire of 5×10⁹ when grown onselective media (designated EBY100 pTQ5-HC*/BJ5457 pTQ6-LC*). Ten cloneswere picked and tested by yeast colony PCR (Example 11) for the presenceof LC and HC containing vectors, and all gave the expected LC and HCproduct. To determine the fraction of the diploid repertoire EBY100pTQ5-HC*/BJ5457 pTQ6-LC* that displayed a HC product and also showedbinding to the antigen streptavidin, a yeast whole cell ELISA wasperformed (Example 7). 68% (15/22) diploids tested displayed a HC, and18% (4/22) of diploid clones tested showed binding to streptavidin.

[0293] In order to highlight the versatility of the procedure, similarhierarchical mating experiments were performed where either thewild-type HC or the wild-type LC was kept constant while varying onlythe corresponding opposite chain. Using the anti-streptavidin F2 Fab asthe model antibody, a diploid repertoire was made from matingEBY100-pTQ5-F2HC and BJ5457 pTQ6-LC*. This diploid repertoire has aconstant HC and variable LC. The mating resulted in 100% of diploidsdisplaying a HC and 30% showing antigen binding by yeast whole cellELISA. Similarly, a diploid repertoire was made by mating BJ5457pTQ6-F2LC with EBY100 pTQ5-HC*. This diploid repertoire has a constantLC and a variable HC. This mating resulted in 70% of diploids displayinga HC and 45% showing antigen binding activity by yeast whole cell ELISA.

EXAMPLE 23 Affinity Selection of a Combinatorially Assembled FabRepertoire

[0294] To demonstrate that a repertoire of yeast cells displaying aplurality of combinatorially assembled Fab antibodies diversified byerror prone PCR can be affinity selected, a combination of selection byKingfisher and affinity driven flow cytometric sorting was used torecover the optimum affinity clones.

[0295] An overnight culture of the diploid repertoire EBY100pTQ5-HC*/BJ5457 pTQ6-LC* (Example 22) was prepared (Example 18). Theculture was induced as in Example 18 and a total of 100 cells weresubjected to one round of Kingfisher selection (Example 7). The antigenbinding yeast diploid cells were retrieved and subjected to FACSaffinity driven selection (see Example 20). The percentage of antigenbinding clones increased during selection as determined by yeast wholecell ELISA (Example 7). The percentage of antigen binding clone alsoincreased, and the antigen mean fluorescent intensity as determined byFACS increased during selection (Table 9). TABLE 9 Selection of matedLC/HC error prone repertoire by combination of Kingfisher and FACS. % %Ag % bind- bind- Ag Input Output cells ing ing Ag Round Conc cells cellsgated ELISA FACS MFI R0 — — — — 18% 2.5% 1.46 R1 beads 10¹⁰ 5 × 10⁶ na85%  35% 2.99 R2 6 nM 10⁷      10⁵ 1.3% 68% 32.2%  7.62 R3 2 nM 10⁶  7,500 0.7% ND ND ND

[0296] The progress of the selection campaign was monitored usingpolylconal FACS analysis where an overnight culture of the selectedrepertoires from each round of selection was prepared and antibodyexpression was induced as in Example 18. Yeast cells were labeled as inExample 20 and analyzed by FACS for both LC display (FITC label) andantigen binding (PE label).

[0297] Selected clones were sequenced and the mutations in the variableLC and variable HC are shown in Table 10. The affinity of selected Fabswas determined using FACS by either an off rate screening assay (Example10) or by a non-linear least squares analysis (data not shown). TABLE 10Analysis of Fab antibodies selected from combinatorial library generatedby yeast mating. HC FACS Factor FACS Factor Clone LC Mutations Mutationsk_(d) e-4s-1 increase K_(d) (nM) increase wt-F2 — — 2.4 1 48 1 R2-12 wtS19F 3 0.8 29 1.6 R2-11 T5A;H34R Q3R;Q77L 1 2.4 23 2.1 R3-6 wtN32K;I69V;Q101V 2.1 1.1 14 3.4 R3-9 wt H53R;Q3R;G31R 1.1 2.2 20 2.4 R3-1wt G8S;S54R;T68S — — — — R3-4 T24S;H34R wt — — — — R3-2 H34R;L95H; wt1.6 1.5 35 1.4 Q79H R3-7 H34R;D32Y; A23D 0.7 3.4 17 2.8 P59S;T69S; A74TR3-8 S27G;T76A H53R — — — —

EXAMPLE 24 Reshuffling of Selected Pools of LC and HC

[0298] To demonstrate the versatility of the procedure and the abilityto do recursive cycles of selection and reshuffling, pools of selectedLC and HC from the output of the third round of selection (Example 23)of the combinatorial EBY100 pTQ5-HC*/BJ5457 pTQ6-LC* repertoire werereshuffled.

[0299] Plasmid DNA was prepared using a lyticase treatment (Example 11),and the DNA extract containing both pTQ5-HC*^(sel) and pTQ6-LC*^(sel)expression plasmids containing selected LC and HC was transformeddirectly into fresh EBY100 and BJ5457 cells, respectively. Thetransformation mix was grown on selective plates so onlyBJ5457-pTQ6-LC*^(sel) colonies (selective agar plates containingblastocidin) or EBY100 pTQ5-HC*^(sel) (selective agar plates minustryptophan) could grow. The BJ5457 pTQ6-LC*^(sel) transformation gave250 colonies and the EBY100 pTQ5-HC*^(sel) transformation gave 25colonies. These two mini-repertoires were harvested and grown overnightand mated as in Example 18. This mating reaction gave a diploidrepertoire of EBY100 pTQ5-HC*^(sel)/BJ5457 pTQ6-LC*^(sel) that coveredthe theoretical combinatorial diversity of 6250 different LC/HCcombinations. Fab antibody expression was induced in the diploid cultureand was selected using AutoMACS. This represented the fourth round ofselection. Diploid culture from the fourth round of selection wasretrieved. Antibody expression was induced, followed by labeling withstreptavidin PE at 0.5 nM and selection using FACS (Example 20). TABLE11 Analysis of Fab antibodies. HC FACS Factor FACS Factor Clone LCMutations Mutations k_(d) e-4s-1 increase K_(d) (nM) increase wt-F2 — —2.4 1 48 1 R5-1 H34R;D32Y;P59S;T69S N32K;I69V; 0.8 2.9 4.2 11.5 A74TQ101V R5-12 H34R;D32Y;P59S;T69S S19F 1 2.4 11.5 4.2 A74T

EXAMPLE 25 Construction of a Naïve HC Repertoire Yeast Display Vectorand Haploid Host Cell

[0300] To produce a novel heavy chain eukaryotic display vector usefulas one component of a multi-chain eukaryotic vector set, a naiverepertoire of HC is cloned into the vector pTQ5 (Example 13).

[0301] Antibody HC fragments are isolated from a V gene peripheral bloodlymphocyte source and isolated by antibody PCR methods known in the art.The HC library is captured in a phage display vector followingtechniques known in the art and then transferred to pTQ5 as a SfiI/NotIfragment and transformed into E. coli, producing a library ofapproximately 1×10⁸. The library is then transformed into yeast strainEBY100, producing library EBY100 pTQ5-HC*^(rep) of approximately 1×10⁷.

Example 26 Construction of a Naïve LC Repertoire Yeast Display Vectorand Haploid Host Cell

[0302] To produce a novel light chain eukaryotic display vector usefulas one component of a multi-chain eukaryotic vector set, a naiverepertoire of LC is cloned into the vector pTQ6 (Example 16).

[0303] Antibody LC fragments are isolated from a V gene peripheral bloodlymphocyte source and isolated by antibody PCR methods known in the art.The LC library is captured in a phage display vector followingtechniques known in the art and then transferred to pTQ6 as a ApaLI/AscIfragment and transformed into E. coli, producing a library ofapproximately 1×10⁸. The library is then transformed into yeast strainW303, producing library W303 pTQ6-LC*rep of approximately 1×10⁷.

Example 27 A LC/HC Recombination Library via Cellular Fusion of aHaploid Host Cell Pair and Subsequent Affinity Selection: Detection byFlow Cytometry

[0304] To produce a novel Fab (diploid) yeast display library two(haploid) host cell populations; one population containing a repertoireof light chain fragments and the second population containing arepertoire of heavy chain fragments, are co-cultured under conditionssufficient to permit cellular fusion and the resulting diploidpopulation grown under conditions sufficient to permit expression anddisplay of the recombined Fab (LC/HC) library.

[0305] Approximately 10¹⁰ EBY100 pTQ5-HC*_(rep) yeast cells (fromExample 26) are mated with approximately 10¹⁰ W303 pTQ6-LC*^(rep) yeastcells (from Example 22) following the procedures outlined in Example 18.Ten percent mating efficiency results in an approximately 10⁹ diploidrepertoire (thus capturing approximately 109 LC/HC combinations of thepossible maximum 10¹⁴ combinatorial LC/HC diversity, given the startingdiversity of the individual component LC and HC repertoires in thehaploid parents). The diploid repertoire is cultured and expression ofLC and HC induced (Example 15). The diploid repertoire is cultured andexpression of LC and HC induced (see Example 15). The diploid culture isincubated with streptavidin-FITC and affinity selected using flowcytometric sorting (see Example 8). Affinity variants are screened byoff rate determination using flow cytometry (see Example 9) andadditionally by surface plasmon resonance techniques known in the art,using soluble Fab antibodies.

Example 28 Production of a Multi-Chain Display Host Cell Pair Library:LC and HC Haploid Yeast Cell Repertoires via Diploid Sporulation

[0306] As one example of a novel host cell pair library, wherein onecell population expresses a plurality of variants of one chain of abiologically active multi-chain polypeptide linked to an anchor protein;and the second cell expresses a plurality of variants of a solublesecond chain of the multi-chain polypeptide, diploid Fab-displayingyeast isolates resulting from the streptavidin selection screen asdescribed in Example 23 are induced to sporulate by culturing theisolates under conditions of nitrogen starvation (as described inGuthrie and Fink, 1991). Sporulated diploids are harvested, treated withzymolase, sonicated, and plated out on rich plates.

[0307] Haploid colonies are separated into two subsamples; one subsampleis grown under conditions to facilitate loss of the LC expression vectorbut selected for the HC display vector, the second subsample is grownunder conditions to facilitate loss of the HC display vector butselected for the LC expression vector (for 2μ derived plasmids under nonselective conditions, plasmid loss is between 2-6% per generation).After several generations each yeast subculture is effectively purged ofnon-selected chain expressing vector and contains only the selected (LCor HC) expression vector, thus producing two biased (i.e., pre-selected)single chain expression haploid yeast cells, designated “HAPLOIDpTQ6-LC*^(sel)” and “HAPLOID pTQ5-HC*^(sel)”. From these two haploidyeast populations, each containing either the light chain ofpre-selected Fabs or the heavy chain of pre-selected Fabs, three matingregimes are established as follows:

[0308] In the first mating regime, 10⁹ yeast HAPLOID pTQ6-LC*^(sel) aremated back with 10⁹ yeast EBY100 pTQ5-HC*^(rep) (from Example 21), andgrown under selective conditions for maintenance of both LC and HC yeastexpression plasmids. Ten percent mating efficiency results inapproximately 10⁸ diploids. The diploid repertoire is cultured andexpression of LC and HC induced (Example 18). The resulting diploidculture represents a biased repertoire containing unique combinations ofthe original HC repertoire against the preselected LC repertoire, whichcan be further screened by, e.g., flow cytometric sorting (Examples 8and 11) and/or surface plasmon resonance techniques known in the art,using soluble Fab antibodies.

[0309] In the second mating regime, 10⁹ yeast HAPLOID pTQ6-HC*^(sel) aremated back with 10⁹ yeast W303 pTQ6-LC*^(rep) (Example 22), and grownunder selective conditions for maintenance of both LC and HC yeastexpression plasmids. Ten percent mating efficiency results inapproximately 10⁸ diploids. The diploid repertoire is cultured andexpression of LC and HC induced (Example 18). The resulting diploidculture represents a biased repertoire containing unique combinations ofthe original LC repertoire against the preselected HC repertoire, whichcan be further screened by, e.g., flow cytometric sorting (Examples 8and 11) and/or surface plasmon resonance techniques known in the art,using soluble Fab antibodies.

[0310] Finally, in the third mating regime, 10⁹ yeast HAPLOIDpTQ6-LC*_(sel) are mated with 10⁹ yeast HAPLOID pTQ6-HC*^(sel), andgrown under selective conditions for maintenance of both LC and HC yeastexpression plasmids. Ten percent mating efficiency results inapproximately 10⁸ diploids. The diploid repertoire is cultured andexpression of LC and HC induced (see Example 18). The resulting diploidculture represents a biased recombination repertoire containing uniquecombinations of the preselected LC repertoire against the preselected HCrepertoire, which can be further screened by, e.g., flow cytometricsorting (Examples 8 and 11) and/or surface plasmon resonance techniquesknown in the art, using soluble Fab antibodies.

Example 29 Affinity Maturation by Restriction Based Diversification of aFab Antibody

[0311] To demonstrate the utility of restriction-based diversificationand shuffling of a Fab antibody for affinity maturation using yeastdisplay and selection, a Fab antibody library is prepared from a leadtarget specific Fab where either the whole LC or a fragment of the HC isdiversified using restriction-based cloning. In one preferred method, anantibody library constructed with restriction sites both bracketing theantibody V gene sequence and also internal to the V gene sequence isused to prepare a plurality of antibody gene fragments for cloning andthus leading to the diversification of the lead antibody.

[0312] Lead antibodies isolated from one such antibody library (e.g.,the CJ library set, Dyax Corp., Cambridge, Mass.) can be affinitymatured by this approach. Antibodies comprising, for example, the CJphagemid library have a LC bracketed by a unique ApaL1 and AscIrestriction site and a HC bracketed by a unique SfiI and NotIrestriction site. The HC also contains an internal and unique XbaIrestriction site between the CDR2 and CDR3 sequence.

[0313] To diversify the LC in either a single antigen specific leadantibody or a pool of antigen specific lead antibodies, the Fab antibodygene(s) are first cloned into the yeast display vector pTQ3 as inExample 2, resulting in pTQ3-Fab. A plurality of LC are isolated from aDNA preparation of the CJ phagemid library by restriction digestion withApaL1 and AscI restriction enzymes. pTQ3-Fab is also digested with ApaL1and AscI, and the endogenous LC is replaced by a plurality of LC givingrise to a repertoire pTQ3-LC^(cj-rep). This repertoire is thentransferred into yeast strain EBY100 to give EBY 100 pTQ3-LC^(cj-rep).

[0314] To diversify the V_(H) CDR1-2 in either an antigen specific leadantibody or a pool of antigen specific lead antibodies first the Fabantibody gene(s) are cloned into the yeast display vector pTQ3 as inExample 2 to give pTQ3-Fab. A plurality of V_(H) CDR1-2 fragments areisolated from the CJ phagemid library by restriction digestion with SfiIand XbaI. pTQ3-Fab is also digested with SfiI and XbaI, and theendogenous V_(H) CDR1-2 fragment is replaced by a plurality of V_(H)CDR1-2 fragments, resulting in the repertoire pTQ3-V_(H)CDR1-2^(cj-rep). This repertoire is then transferred into yeast strainEBY100 to give EBY100 pTQ3-V_(H) CDR1-2C^(cj-rep).

[0315] It will be clear to those skilled in the art that the cloningprocedure can be performed in a number of different ways, e.g., by firstconstructing a repertoire of V_(H) CDR1-2 fragments and then cloning inthe antigen specific V_(H) CDR3 or pool of V_(H) CDR3s.

[0316] A culture of EBY100 pTQ3-V_(H) CDR1-2c^(cj-rep) and EBY100pTQ3-LC^(cj-rep) is prepared as in Example 2. The yeast culture is thenlabeled for LC display and antigen binding and affinity selected by flowcytometric sorting as in Example 10. Selected clones are then analyzedfor their DNA sequence and there improvement in affinity as in Example10.

Example 30 Affinity Maturation by Combinatorial Shuffling of GeneFragments Using Yeast Mating

[0317] To demonstrate that yeast mating can be used for combinatorialgene diversification and affinity maturation of an antigen specific leadantibody or antigen specific lead antibodies, a selected LC or pool ofLCs is rediversified or a V_(H) CDR1-2 fragment of a selected HC or poolof HCs is rediversified. Antibodies comprising the CJ phagemid libraryare amenable to such an approach. They have a LC bracketed by a uniqueApaL1 and AscI restriction site and a HC bracketed by a unique SfiI andNotI restriction site. The HC also contains an internal and unique XbaIrestriction site between the CDR2 and CDR3 sequence. As the LC and HCare present in yeast cells of opposite mating type, yeast mating is usedto bring together antigen specific LC with a plurality of V_(H) CDR1-2fragments or antigen specific HC with a plurality of LC, thuseliminating the need for restriction-based cloning to pair a LC with aHC.

[0318] In one preferred method to diversify an antigen specific leadantibody or a pool of antigen specific lead antibodies, the component HCantibody genes are cloned into the yeast display vector pTQ5 as inExample 13 to give pTQ5-HCAg. A plurality of V_(H) CDR1-2 fragments areprepared by digestion of HC fragments from the CJ phagemid library withSfiI and XbaI restriction enzymes. This plurality of V_(H) CDR1-2fragments is then cloned into DNA prepared from pTQ5-HCAg, which hasbeen digested with SfiI and XbaI to remove the endogenous V_(H) CDR1-2fragment and to replace with the plurality of V_(H) CDR1-2 fragments,the antigen specific V_(H)-CDR3 being retained. This gives a librarydesignated pTQ5-V_(H) CDR1-2 (CDR3Ag) and this is introduced into theyeast strain EBY100 to give a repertoire EBY100 PTQ5-V_(H) CDR1-2(CDR3Ag). A plurality of LC are isolated from a DNA preparation of theCJ phagemid library by restriction digestion with ApaL1 and AscI. Thisplurality of LC is cloned into pTQ6 to give a repertoire pTQ6-LC^(rep)and serves as one master repertoire for affinity maturation of otherantibodies specific for other targets. This repertoire is thentransferred into a yeast strain of the opposite mating type, BJ5457, togive BJ5457 pTQ6-LC^(rep).

[0319] In one mating regime, which allows for the simultaneousdiversification of both the LC and the V_(H) CDR1-2 gene fragment,cultures of EBY100 pTQ5-V_(H) CDR1-2 (CDR3Ag) and BJ5457 pTQ6-LC^(rep)are prepared as in Example 22. The two repertoires are mated with eachother (see Example 19) to give a diploid repertoire EBY100 pTQ5-V_(H)CDR1-2 (CDR3Ag)/BJ5457 pTQ6-LC^(rep). Fab antibody expression is induced(see Example 18) and the diploid repertoire is affinity selected as inExample 20. Selected clones are analyzed for improved affinity as inExample 23.

[0320] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details canbe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A multi-chain polypeptide eukaryotic displayvector comprising: (a) a first polynucleotide encoding a polypeptidecomprising a first chain of a biologically active multi-chainpolypeptide linked to a cell surface anchor; and (b) a secondpolynucleotide encoding a second chain of the multi-chain polypeptide;wherein the vector is operable in a eukaryotic host cell to directexpression and secretion of the chains of the multi-chain polypeptide,and wherein the chains of the multi-chain polypeptide associate suchthat the biological activity of the multi-chain polypeptide is exhibitedat the surface of the eukaryotic host cell.
 2. The eukaryotic displayvector of claim 1, further comprising (c) a third polynucleotideencoding a third chain of the multi-chain polypeptide.
 3. The eukaryoticdisplay vector of claim 2, further comprising (d) a fourthpolynucleotide encoding a fourth chain of the multi-chain polypeptide.4. The eukaryotic display vector of claim 1, wherein the multi-chainpolypeptide is a two-chain polypeptide.
 5. The eukaryotic display vectorof claim 1, wherein the multi-chain polypeptide is a four-chainpolypeptide, wherein the four-chain polypeptide is comprised of twofirst chains and two second chains.
 6. The eukaryotic display vector ofclaim 1, wherein the multi-chain polypeptide is a two-chain polypeptideselected from the group consisting of: immunoglobulin Fab fragments, theextracellular domains of T cell receptors, MHC class I molecules and MHCclass II molecules.
 7. The eukaryotic display vector of claim 1, whereinthe multi-chain polypeptide is an immunoglobulin (Ig) or an Ig fragment.8. The eukaryotic display vector of claim 7, wherein the multi-chainpolypeptide is an immunoglobulin selected from the group consisting of:IgA, IgD, IgE, IgG and IgM.
 9. The eukaryotic display vector of claim 8,wherein the multi-chain polypeptide is an IgG.
 10. The eukaryoticdisplay vector of claim 7, wherein the multi-chain polypeptide is aFab.11. The eukaryotic display vector of claim 1, wherein the anchor is acell surface protein of a eukaryotic cell.
 12. The eukaryotic displayvector of claim 1, wherein the anchor is a portion of a cell surfaceprotein of a eukaryotic cell that anchors to the cell surface of theeukaryotic host cell.
 13. The eukaryotic display vector of claim 1,wherein the anchor is selected from the group consisting of:α-agglutinin, a-agglutinin components Aga1p and Aga2p, and FLO1.
 14. Theeukaryotic display vector of claim 1, wherein, on expression, the firstchain and the cell surface anchor are expressed as a fusion protein inthe eukaryotic host cell.
 15. The eukaryotic display vector of claim 1,wherein, on expression, the first chain is linked to the cell surfaceanchor by means of a Jun/Fos linkage.
 16. The eukaryotic display vectorof claim 14, wherein, on expression, the first chain of the multi-chainpolypeptide is fused to Aga2p, wherein Aga2p is covalently linked toAga1p, which in turn is linked to the eukaryotic host cell surface. 17.The eukaryotic display vector of claim 1, wherein the firstpolynucleotide is operably linked to an Aga2p signal sequence and thesecond polynucleotide is operably linked to an Aga2p signal sequence.18. The eukaryotic display vector of claim 1, wherein the firstpolynucleotide is linked in frame to a polynucleotide encoding a firstepitope tag, and the second polynucleotide is linked in frame to apolynucleotide encoding a second epitope tag.
 19. The eukaryotic displayvector of claim 1, wherein the vector further comprises restrictionendonuclease recognition sites located at the 5′ and 3′ ends of apolynucleotide segment including all of the polynucleotides encoding thechains of the multi-chain polypeptide.
 20. The eukaryotic display vectorof claim 1, wherein the vector further comprises restrictionendonuclease recognition sites located at the 5′ and 3′ ends of each ofthe polynucleotides encoding the chains of the multi-chain polypeptide.21. A vector library comprising a eukaryotic display vector according toclaim
 1. 22. The vector library of claim 21, wherein the librarycomprises a heterogeneous population of multi-chain polypeptides.
 23. Amethod of displaying a biologically active multi-chain polypeptide onthe surface of a eukaryotic host cell comprising utilizing the vector ofclaim
 1. 24. The eukaryotic display vector of claim 1, wherein theeukaryotic display vector is a vector selected from the group consistingof: a vector set, a dual display vector, and a yeast display vector. 25.The eukaryotic display vector of claim 24, wherein the multi-chainpolypeptide is a two-chain polypeptide, and the eukaryotic displayvector is a vector set comprising a first eukaryotic vector and a secondeukaryotic vector, each vector comprising a polynucleotide encoding onechain of the two-chain polypeptide.
 26. The eukaryotic display vector ofclaim 24, wherein the eukaryotic display vector is a vector set andwherein the multi-chain polypeptide is a three-chain polypeptide, andthe vector set comprises a first eukaryotic vector, a second eukaryoticvector, and a third eukaryotic vector, each vector comprising apolynucleotide encoding one chain of the three-chain polypeptide. 27.The eukaryotic display vector set of claim 24, wherein the eukaryoticdisplay vector is a vector set and wherein the vector set comprises atleast the following three vectors: (a) a first eukaryotic vectorcomprising a first polynucleotide encoding the first chain of thefour-chain polypeptide linked to a cell surface anchor, wherein thevector is operable in a eukaryotic host cell to direct expression andsecretion of the first chain; (b) a second eukaryotic vector comprisingthe first polynucleotide encoding the first chain of the four-chainpolypeptide, wherein the vector is operable in a eukaryotic host cell todirect expression and secretion of the first chain; and (c) a thirdeukaryotic vector comprising a second polynucleotide encoding the secondchain of the four-chain polypeptide, wherein the vector is operable in aeukaryotic host cell to direct expression and secretion of the secondchain, thereby forming a vector set, wherein a eukaryotic host celltransformed with the vector set, on expression of the first and secondpolynucleotides, exhibits the biological activity of the four-chainpolypeptide at the surface of the eukaryotic host cell.
 28. Theeukaryotic display vector of claim 24, wherein the eukaryotic displayvector is a dual display vector and wherein the anchor is a polypeptideoperable as an anchor on the surface of a eukaryotic cell and operableas an anchor on the surface of a phage.
 29. The dual display vector ofclaim 28, wherein the anchor is a portion of a surface protein thatanchors to the cell surface of a eukaryotic host cell and to the surfaceof a phage.
 30. The eukaryotic display vector of claim 24, wherein theeukaryotic display vector is a dual display vector and wherein the firstpolynucleotide is operably linked to a polynucleotide encoding a firstsignal sequence, and the second polynucleotide is operably linked to apolynucleotide encoding a second signal sequence, wherein the firstsignal sequence and the second signal sequence are operable in abacterial cell and operable in a eukaryotic cell.
 31. The eukaryoticdisplay vector of claim 24, wherein the eukaryotic display vector is adual display vector and wherein the first polynucleotide is operablylinked to a first promoter and the second polynucleotide is operablylinked to a second promoter, wherein the first promoter and the secondpromoter are both operable in a bacterial cell and operable in aeukaryotic cell.
 32. A yeast Fab display vector comprising: (a) a firstpolynucleotide encoding a first polypeptide comprising V_(H) and C_(H)1regions of an Ig heavy chain linked to a cell surface anchor operable inyeast; and (b) a second polynucleotide encoding a second polypeptidecomprising an Ig light chain; wherein the vector is operable in a yeasthost cell to direct expression and secretion of the chains of themulti-chain polypeptide, and wherein the chains of the multi-chainpolypeptide associate to form a Fab at the surface of the yeast hostcell.
 33. The yeast Fab display vector of claim 32, wherein the anchoris selected from the group consisting of: α-agglutinin, a-agglutinincomponents Aga1p and Aga2p, and FLO1.
 34. The yeast Fab display vectorof claim 32, wherein, on expression, the V_(H) and C_(H)1 regions arelinked to the cell surface anchor by means of a Jun/Fos linkage.
 35. Theyeast Fab display vector of claim 33, wherein, on expression, the V_(H)and C_(H)1 regions are fused to Aga2p, and the Aga2p is covalentlylinked to Aga1p, which in turn is linked to the yeast host cell surface.36. The yeast Fab display vector of claim 32, wherein the vector furthercomprises restriction endonuclease recognition sites located at the 5′and 3′ ends the polynucleotides encoding the chains of the multi-chainpolypeptide.
 37. The Fab yeast display vector of claim 32, wherein thevector is a vector set.
 38. The Fab yeast display vector of claim 32,wherein the vector is a dual display vector.
 39. The Fab yeast displayvector of claim 38, wherein the dual display vector is operable in phageand yeast.
 40. A Fab yeast display library comprising a yeast displayvectors according to claim
 32. 41. The Fab yeast display library ofclaim 40, wherein the yeast display library comprises a heterogeneouspopulation of multi-chain polypeptides.
 42. A method of displaying abiologically active multi-chain polypeptide on the surface of aeukaryotic host cell comprising utilizing one or more vectors of claim32.
 43. A method for displaying, on the surface of a eukaryotic hostcell, a biologically active multi-chain polypeptide comprising at leasttwo polypeptide chains, the process comprising the steps of: (a)introducing into a eukaryotic host cell (i) a first eukaryotic vectorcomprising a first polynucleotide encoding a first polypeptide chain ofa biologically active multi-chain polypeptide linked to a cell surfaceanchor, wherein the vector is operable in a eukaryotic host cell todirect expression and secretion of the first chain; and (ii) a secondeukaryotic vector comprising a second polynucleotide encoding a secondpolypeptide chain of the multi-chain polypeptide, wherein the vector isoperable in a eukaryotic host cell to direct expression and secretion ofthe second chain,  wherein a eukaryotic host cell transformed with thefirst eukaryotic vector and the second eukaryotic vector exhibits, onexpression of the first and second polynucleotides, the biologicalactivity of the multi-chain polypeptide at the surface of the eukaryotichost cell; and (b) culturing the host cell under conditions suitable forexpression of the first and second polynucleotides.
 44. The method ofclaim 43, wherein the eukaryotic host cell is selected from the groupconsisting of: an animal cell, a plant cell, and a fungus cell.
 45. Themethod of claim 43, wherein the eukaryotic host cell is selected fromthe group consisting of: a mammalian cell, an insect cell, and a yeastcell.
 46. The method of claim 43, wherein the eukaryotic host cell is ayeast cell.
 47. The method of claim 46, wherein the yeast cell is of agenus selected from the group consisting of: Saccharomyces, Pichia,Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, Debaryomycesand Candida.
 48. The method of claim 47, wherein the yeast cell isselected from the group consisting of: Saccharomyces cerevisiae,Hansenula polymorpha, Kluyveromyces lactis, Pichia pastoris,Schizosaccharomyces pombe and Yarrowia lipolytica.
 49. The method ofclaim 47, wherein the yeast cell is Saccharomyces cerevisiae.
 50. Amethod of displaying a biologically active multi-chain polypeptidecomprising at least two polypeptide chains on the surface of a diploideukaryotic cell, comprising: (a) providing a first haploid eukaryoticcell comprising a first polynucleotide encoding a polypeptide comprisinga first chain of a biologically active multi-chain polypeptide linked toa cell surface anchor; (b) providing a second haploid eukaryotic cell,wherein the second haploid eukaryotic cell comprises a secondpolynucleotide encoding a polypeptide comprising a second chain of themulti-chain polypeptide; (c) contacting the first haploid eukaryoticcell with the second haploid eukaryotic cell under conditions sufficientto permit the cells to fuse, producing a diploid eukaryotic cell; and(d) culturing the diploid eukaryotic cell under conditions sufficient topermit expression and association of the chains of the multi-chainpolypeptide, wherein the biological activity of the multi-chainpolypeptide is exhibited at the surface of the diploid eukaryotic cell.51. The method of claim 50, wherein the first haploid eukaryotic celland the second haploid eukaryotic cell are of opposite mating type. 52.The method of claim 50, wherein the first haploid eukaryotic cell andthe second haploid eukaryotic cell are of opposite mating type.
 53. Amethod for displaying a Fab on the surface of a diploid yeast cellcomprising the steps of: (a) providing a first haploid yeast cellcomprising a first polynucleotide encoding a first polypeptidecomprising V_(H) and C_(H)1 regions of an Ig heavy chain linked to acell surface anchor; (b) providing a second haploid yeast cell, whereinthe second haploid yeast cell comprises a second polynucleotide encodinga second polypeptide comprising an Ig light chain; (c) contacting thefirst haploid yeast cell with the second haploid yeast cell underconditions sufficient to permit the cells to fuse, producing a diploidyeast cell; and (d) culturing the diploid yeast cell under conditionssufficient to permit expression and association of the first and secondpolypeptides, wherein a Fab is exhibited at the surface of the diploidyeast cell.
 54. The method of claim 53, wherein the first haploid yeastcell and the second haploid yeast cell are of opposite mating type. 55.The method of claim 54, wherein the first and second haploid yeast cellsare cells of Saccharomyces cerevisiae.
 56. A method for detecting abiologically active multi-chain polypeptide comprising at least twopolypeptide chains from a multi-chain polypeptide library, comprising:(a) providing a first haploid eukaryotic cell population comprising aplurality of first polynucleotides each encoding a polypeptidecomprising a first chain variant of a biologically active multi-chainpolypeptide linked to a cell surface anchor; (b) providing a secondhaploid eukaryotic cell population of opposite mating type to that ofthe first haploid eukaryotic cell population, wherein the second haploideukaryotic cell population comprises a plurality of secondpolynucleotides each encoding a second chain variant of the multi-chainpolypeptide; (c) contacting the first haploid eukaryotic cell populationwith the second haploid eukaryotic cell population under conditionssufficient to permit individual cells of different mating types to fuse,producing a population of diploid eukaryotic cells; (d) culturing thediploid eukaryotic cells under conditions sufficient to permitexpression and association of the chains of the multi-chain polypeptide,wherein the biological activity of the multi-chain polypeptide isexhibited at the surface of the diploid eukaryotic cells; and (e)detecting a particular biological activity of interest.
 57. The methodof claim 56, further comprising the step: (f) isolating diploideukaryotic cells that display the biological activity.
 58. The method ofclaim 57, further comprising the steps of: (g) repeating steps (d), (e)and (f).
 59. The method of claim 57, further comprising the steps of:(g) culturing the isolated diploid eukaryotic cells of step (f) underconditions sufficient to cause the isolated diploid eukaryotic cells toundergo meiosis, producing haploid eukaryotic cells; (h) contacting thehaploid eukaryotic cells from step (g) under conditions sufficient topermit eukaryotic cells of different mating types to fuse, producing apopulation of diploid eukaryotic cells; and (i) repeating steps (d), (e)and (f).
 60. The method of claim 57, further comprising the steps of:(g) culturing the isolated diploid eukaryotic cells of step (f) underconditions sufficient to cause the isolated diploid eukaryotic cells toundergo meiosis, producing haploid eukaryotic cells; (h) contacting thehaploid eukaryotic cells from step (g) with one or more haploideukaryotic cell populations selected from the group consisting of: (i)the first haploid eukaryotic cell population of step (a); (ii) thesecond haploid eukaryotic cell population of step (b); (iii) a thirdhaploid eukaryotic cell population of opposite mating type to that ofthe second haploid eukaryotic cell population, wherein the third haploideukaryotic cell population comprises a plurality of firstpolynucleotides encoding a polypeptide comprising a first chain variantof a biologically active multi-chain polypeptide linked to a cellsurface anchor; and (iv) a fourth haploid eukaryotic cell population ofopposite mating type to that of the first haploid eukaryotic cellpopulation, wherein the fourth haploid eukaryotic cell populationcomprises a plurality of second polynucleotides each encoding a secondchain variant of the multi-chain polypeptide, under conditionssufficient to permit eukaryotic cells of different mating types to fuse,producing a population of diploid eukaryotic cells; and (i) repeatingsteps (d), (e) and (f).
 61. A method for detecting and isolating one ormore multi-chain polypeptides that exhibit a biological activity ofinterest comprising: (a) providing a eukaryotic cell wherein, onexpression of a multi-chain eukaryotic display vector, the eukaryoticcell displays a multi-chain polypeptide on its surface; (b) culturingthe eukaryotic cell under conditions sufficient to permit expression ofthe multi-chain polypeptide; and (c) contacting the cells with amolecule of interest; and (d) selecting and isolating cells that exhibita particular interaction with the molecule of interest.
 62. The methodof claim 61, wherein a host cell displaying the multi-chain polypeptideexhibiting the biological activity of interest is isolated, and,optionally, is subjected to at least one additional round of selection.63. The method of claim 61, further comprising screening the libraryusing a phage display screen.
 64. The method of claim 61, wherein themolecule of interest is a protein.
 65. The method of claim 64, whereinthe biological activity of interest is an interaction between themulti-chain polypeptide and another molecular species and comprises anon-covalent association between the molecular species.
 66. The methodof claim 65, wherein the interaction is transient.
 67. The method ofclaim 65, wherein the interaction is a covalent interaction.
 68. Amethod for transferring nucleic acid sequences encoding a biologicallyactive multi-chain polypeptide between a phage display vector and aeukaryotic display vector comprising: (a) obtaining a phage displayvector comprising: (i) a first polynucleotide encoding a polypeptidecomprising a first chain of the biologically active multi-chainpolypeptide, wherein the first chain is linked to a cell surface anchor,and (ii) a second polynucleotide encoding a second chain of themulti-chain polypeptide,  wherein the phage display vector is operablein a bacterial host cell to direct expression of the chains of themulti-chain polypeptide, and wherein the chains of the multi-chainpolypeptide associate such that the biological activity of themulti-chain polypeptide is exhibited at the surface of a phagecomprising the phage display vector and propagating in the bacterialhost cell; and (b) inserting the first and second polynucleotidesencoding the chains of the multi-chain polypeptide into a eukaryoticdisplay vector, wherein the eukaryotic display vector is operable in aeukaryotic host cell to direct expression and secretion of the chains ofthe multi-chain polypeptide, and wherein the chains of the multi-chainpolypeptide associate such that the biological activity of themulti-chain polypeptide is exhibited at the surface of the eukaryotichost cell.
 69. The method of claim 68, wherein the polynucleotidesequences encoding each of the chains of the multi-chain polypeptide areinserted together as a single polynucleotide into the eukaryotic displayvector.
 70. The method of claim 68, wherein the polynucleotide sequencesencoding each of the chains of the multi-chain polypeptide areindependently inserted as separate polynucleotides into the eukaryoticdisplay vector.
 71. The method of claim 68, wherein the transferringstep (b) comprises a genetic transfer technique selected from the groupconsisting of: restriction digestion, PCR amplification, homologousrecombination and combinations of such techniques.
 72. A method fortransferring nucleic acid sequences encoding a biologically active Fabbetween a phage display vector and a eukaryotic display vectorcomprising: (a) obtaining a phage display vector comprising: (i) a firstpolynucleotide encoding a first polypeptide comprising V_(H) and C_(H)1regions of an Ig heavy chain linked a cell surface anchor, and (ii) asecond polynucleotide encoding a second polypeptide comprising an Iglight chain,  wherein the phage display vector is operable in abacterial host cell to direct expression of the first and secondpolypeptides, and wherein the polypeptides associate such that thebiological activity of the multi-chain polypeptide is exhibited at thesurface of a phage transfected with the phage display vector andpropagating in the bacterial host cell; and (b) inserting the first andsecond polynucleotides encoding the first and second polypeptides into aeukaryotic display vector, wherein the eukaryotic display vector isoperable in a yeast host cell to direct expression and secretion of thefirst and second polypeptides, and wherein the polypeptides associatesuch that the biological activity of the Fab is exhibited at the surfaceof the yeast host cell.
 73. The method of claim 72, wherein thepolynucleotide sequences encoding each of the polypeptides are insertedtogether as a single polynucleotide into the eukaryotic display vector.74. The method of claim 72, wherein the polynucleotide sequencesencoding each of the polypeptides are independently inserted as separatepolynucleotides into the eukaryotic display vector.
 75. The method ofclaim 72, wherein the transferring step (b) comprises a genetic transfertechnique selected from the group consisting of restriction digestion,PCR amplification, homologous recombination, and combinations of suchtechniques.
 76. A eukaryotic host cell comprising a vector comprising:(a) a first polynucleotide encoding a polypeptide comprising a firstchain of a biologically active multi-chain polypeptide linked to a cellsurface anchor; and (b) a second polynucleotide encoding a second chainof the multi-chain polypeptide; wherein the vector is operable in aeukaryotic host cell to direct expression and secretion of the chains ofthe multi-chain polypeptide, and wherein the chains of the multi-chainpolypeptide associate such that the biological activity of themulti-chain polypeptide is exhibited at the surface of the eukaryotichost cell.
 77. The eukaryotic host cell of claim 76, wherein theeukaryotic host cell is selected from the group consisting of: an animalcell, a plant cell, and a fungus cell.
 78. The eukaryotic host cell ofclaim 76, wherein the eukaryotic host cell is selected from the groupconsisting of: a mammalian cell, an insect cell, and a yeast cell. 79.The eukaryotic host cell of claim 76, wherein the eukaryotic host cellis a yeast cell.
 80. The eukaryotic host cell of claim 79, wherein theyeast cell is haploid.
 81. The eukaryotic host cell of claim 79, whereinthe yeast cell is diploid.
 82. A pair of haploid eukaryotic cellscomprising: (a) a first haploid eukaryotic cell comprising a firstpolynucleotide encoding a polypeptide comprising a first chain of abiologically active multi-chain polypeptide linked to a cell surfaceanchor; and (b) a second haploid eukaryotic cell comprising a secondpolynucleotide encoding a second chain of the multi-chain polypeptide.83. The haploid eukaryotic cell pair of claim 82, wherein the firsthaploid eukaryotic cell and the second haploid eukaryotic cell are ofopposite mating type.
 84. The haploid eukaryotic cell pair of claim 82,wherein the multi-chain polypeptide is a two-chain polypeptide.
 85. Thehaploid eukaryotic cell pair of claim 82, wherein the multi-chainpolypeptide is a four-chain polypeptide and wherein the four-chainpolypeptide is comprised of two the first chains and two the secondchains.
 86. The haploid eukaryotic cell pair of claim 82, wherein themulti-chain polypeptide is a two-chain polypeptide selected from thegroup consisting of immunoglobulin Fab fragments, and the extracellulardomains of T cell receptors, MHC class I molecules, and MHC class IImolecules.
 87. The haploid eukaryotic cell pair of claim 82, wherein theanchor is a cell surface protein of a eukaryotic cell.
 88. A eukaryotichost cell library comprising a plurality of diploid cells that are thefusion product of a plurality of eukaryotic host cell pairs according toclaim
 82. 89. The eukaryotic host cell library of claim 88, wherein theplurality of diploid cells display a heterogeneous population ofmulti-chain polypeptides.
 90. A yeast cell transformed with aheterologous display vector comprising: (a) a first polynucleotideencoding a polypeptide comprising a first chain of a biologically activemulti-chain polypeptide linked to a cell surface anchor operable inyeast; and (b) a second polynucleotide encoding a second chain of themulti-chain polypeptide; wherein the vector is operable in the yeastcell to direct expression and secretion of the chains of the multi-chainpolypeptide, and wherein the chains of the multi-chain polypeptideassociate such that the biological activity of the multi-chainpolypeptide is exhibited at the surface of the yeast cell.
 91. A pair ofhaploid yeast cells comprising: (a) a first haploid yeast cellcomprising a first polynucleotide encoding a polypeptide comprising afirst chain of a biologically active multi-chain polypeptide linked to acell surface anchor; and (b) a second haploid yeast cell comprising asecond polynucleotide encoding a second chain of the multi-chainpolypeptide.
 92. The haploid yeast cell pair of claim 91, wherein thefirst haploid yeast cell and the second haploid yeast cell are ofopposite mating type.
 93. A yeast display library comprising apopulation of yeast cells collectively displaying a repertoire of atleast 10⁷ polypeptides.
 94. A yeast display library comprising apopulation of yeast cells collectively displaying a heterogeneouspopulation of at least 104 multi-chain polypeptides.
 95. The yeastdisplay library of claim 94, comprising a population of yeast cellscollectively displaying a heterogeneous population of at least 10⁷multi-chain polypeptides.
 96. The yeast display library of claim 95,comprising a population of yeast cells collectively displaying aheterogeneous population of at least 10⁸ multi-chain polypeptides.
 97. Ayeast display library comprising a plurality of diploid cells that arethe fusion product of a plurality of yeast cell pairs according to claim91.
 98. The yeast display library of claim 97, wherein the plurality ofdiploid cells display a heterogeneous population of Fabs.